EP1037977B1 - ADNc PROLONGES POUR PROTEINES SECRETEES - Google Patents

ADNc PROLONGES POUR PROTEINES SECRETEES Download PDF

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EP1037977B1
EP1037977B1 EP98959117A EP98959117A EP1037977B1 EP 1037977 B1 EP1037977 B1 EP 1037977B1 EP 98959117 A EP98959117 A EP 98959117A EP 98959117 A EP98959117 A EP 98959117A EP 1037977 B1 EP1037977 B1 EP 1037977B1
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sequences
sequence
extended
protein
cdna
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EP1037977A2 (fr
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Lydie Bougueleret
Aymeric Duclert
Jean-Baptiste Dumas Milne Edwards
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Merck Biodevelopment SAS
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Serono Genetics Institute SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the estimated 50.000-100.000 genes scattered along the human chromosomes offer tremondous promise for the understanding, diagnosis, and treatment of human diseases.
  • probes capable of specifically hybridizing to loci distributed throughout the human genome find applications in the construction of high resolution chromosome maps and in the identification of individuals.
  • Cloning vectors such as yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) are able to accept DNA inserts ranging from 300 to 1000 kilobases (kb) or 100-400 kb in length respectively, thereby facilitating the manipulation and ordering of DNA sequences distributed over great distances on the human chromosomes.
  • Automated DNA sequencing machines permit the rapid sequencing of human genes. Bioinformatics software enables the comparison of nucleic acid and protein sequences, thereby assisting in the characterization of human gene products.
  • cDNAs complementary DNAs
  • mRNAs messenger RNAs
  • sequencing is only performed on DNA which is derived from protein coding portions of the genome.
  • ESTs expressed sequence tags
  • the ESTs may then be used to isolate or purify extended cDNAs which include sequences adjacent to the EST sequences.
  • the extended cDNAs may contain all of the sequence of the EST which was used to obtain them or only a portion of the sequence of the EST which was used to obtain them.
  • the extended cDNAs may contain the full coding sequence of the gene from which the EST was derived or, alternatively, the extended cDNAs may include portions of the coding sequence of the gene from which the EST was derived. It will be appreciated that there may be several extended cDNAs which include the EST sequence as a result of alternate splicing or the activity of alternative promoters.
  • the short EST sequences which could be used to isolate or purify extended cDNAs were often obtained from oligo-dT primed cDNA libraries. Accordingly, they mainly corresponded to the 3' untranslated region of the mRNA. In part, the prevalence of EST sequences derived from the 3' end of the mRNA is a result of the fact that typical techniques for obtaining cDNAs, are not well suited for isolating cDNA sequences derived from the 5' ends of mRNAs. ( Adams et al., Nature 377:174, 1996 , Hillier et al., Genome Res. 6:807-828, 1996 ).
  • the reported sequences typically correspond to coding sequences and do not include the full 5' untranslated region of the mRNA from which the cDNA is derived.
  • Such incomplete sequences may not include the first exon of the mRNA, particularly in situations where the first exon is short.
  • they may not include some exons, often short ones, which are located upstream of splicing sites.
  • sequences derived from the 5' ends of mRNAs which can be used to obtain extended cDNAs which may include the 5' sequences contained in the 5' ESTs.
  • secretory proteins including tissue plasminogen activator, G-CSF, GM-CSF, erythropoietin, human growth hormone, insulin, interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , and interleukin-2.
  • tissue plasminogen activator G-CSF
  • GM-CSF GM-CSF
  • erythropoietin human growth hormone
  • insulin interferon- ⁇
  • interferon- ⁇ interferon- ⁇
  • interferon- ⁇ interleukin-2
  • interleukin-2 interleukin-2
  • secretory proteins include short peptides, called signal peptides, at their amino termini which direct their secretion.
  • signal peptides are encoded by the signal sequences located at the 5' ends of the coding sequences of genes encoding secreted proteins. Because these signal peptides will direct the extracellular secretion of any protein to which they are operably linked, the signal sequences may be exploited to direct the efficient secretion of any protein by operably linking the signal sequences to a gene encoding the protein for which secretion is desired. This may prove beneficial in gene therapy strategies in which it is desired to deliver a particular gene product to cells other than the cell in which it is produced.
  • Signal sequences encoding signal peptides also find application in simplifying protein purification techniques. In such applications, the extracellular secretion of the desired protein greatly facilitates purification by reducing the number of undesired proteins from which the desired protein must be selected. Thus, there exists a need to identify and characterize the 5' portions of the genes for secretory proteins which encode signal peptides.
  • 5' ESTs and extended cDNAs obtainable therefrom may be used to efficiently identify and isolate upstream regulatory regions which control the location, developmental stage, rate, and quantity of protein synthesis, as well as the stability of the mRNA. ( Theil et al., BioFactors 4:87-93, (1993 ). Once identified and characterized, these regulatory regions may be utilized in gene therapy or protein purification schemes to obtain the desired amount and locations of protein synthesis or to inhibit, reduce, or prevent the synthesis of undesirable gene products.
  • ESTs containing the 5' ends of secretory protein genes or extended cDNAs which include sequences adjacent to the sequences of the ESTs may include sequences useful as probes for chromosome mapping and the identification of individuals.
  • the purified, isolated or recombinant extended cDNAs may contain the entire open reading frame of their corresponding mRNAs, including a start codon and a stop codon.
  • the extended cDNAs may include nucleic acids encoding the signal peptide as well as the mature protein.
  • the extended cDNAs may contain a fragment of the open reading frame.
  • the fragment may encode only the sequence of the mature protein.
  • the fragment may encode only a portion of the mature protein.
  • a nucleic acid which encodes the signal peptide of a secreted protein.
  • the present extended cDNAs were obtained using ESTs which include sequences derived from the authentic 5' ends of their corresponding mRNAs.
  • EST or "5' EST” refer to the short cDNAs which were used to obtain the extended cDNAs of the present invention.
  • extended cDNA refers to the cDNAs which include sequences adjacent to the 5' EST used to obtain them.
  • the extended cDNAs may contain all or a portion of the sequence of the EST which was used to obtain them.
  • corresponding mRNA refers to the mRNA which was the template for the cDNA synthesis which produced the 5' EST.
  • the term "purified" does not require absolute purity; rather, it is intended as a relative definition.
  • Individual extended cDNA clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. The sequences obtained from these clones could not be obtained directly either from the library or from total human DNA.
  • the extended cDNA clones are not naturally occurring as such, but rather are obtained via manipulation of a partially purified naturally occurring substance (messenger RNA).
  • the conversion of mRNA into a cDNA library involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection.
  • creating a cDNA library from messenger RNA and subsequently isolating individual clones from that library results in an approximately 10 4 -10 6 fold purification of the native message.
  • Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • isolated requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • the term “recombinant” means that the extended cDNA is adjacent to "backbone" nucleic acid to which it is not adjacent in its natural environment. Additionally, to be “enriched” the extended cDNAs will represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid backbone molecules.
  • Backbone molecules according to the present invention include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest.
  • the enriched extended cDNAs represent 15% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules.
  • the enriched extended cDNAs represent 50% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules. In a highly preferred embodiment, the enriched extended cDNAs represent 90% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules. "Stringent”, “moderate,” and “low” hybridization conditions are as defined in Example 29.
  • extended cDNAs encoding secreted polypeptides or fragments thereof which are present in cDNA libraries in which one or more extended cDNAs encoding secreted polypeptides or fragments thereof make up 5% or more of the number of nucleic acid inserts in the backbone molecules are "enriched recombinant extended eDNAs" as defined herein.
  • extended cDNAs encoding secreted polypeptides or fragments thereof which are in a population of plasmids in which one or more extended cDNAs of the present invention have been inserted such that they represent 5% or more of the number of inserts in the plasmid backbone are " enriched recombinant extended cDNAs" as defined herein.
  • extended cDNAs encoding secreted polypeptides or fragments thereof which are in cDNA libraries in which the extended cDNAs encoding secreted polypeptides or fragments thereof constitute less than 5% of the number of nucleic acid inserts in the population of backbone molecules, such as libraries in which backbone molecules having a cDNA insert encoding a secreted polypeptide are extremely rare, are not "enriched recombinant extended cDNAs.”
  • a "secreted” protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal peptides in its amino acid sequence.
  • "Secreted” proteins include without limitation proteins secreted wholly (e.g. soluble proteins), or partially (e.g. receptors) from the cell in which they are expressed.
  • “Secreted” proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.
  • Extended cDNAs encoding secreted proteins may include nucleic acid sequences, called signal sequences, which encode signal peptides which direct the extracellular secretion of the proteins encoded by the extended cONAs. Generally, the signal peptides are located at the amino termini of secreted proteins.
  • Secreted proteins are translated by ribosomes associated with the "rough" endoplasmic reticulum. Generally, secreted proteins are co-translationally transferred to the membrane of the endoplasmic reticulum. Association of the ribosome with the endoplasmic reticulum during translation of secreted proteins is mediated by the signal peptide. The signal peptide is typically cleaved following its co-translational entry into the endoplasmic reticulum. After delivery to the endoplasmic reticulum, secreted proteins may proceed through the Golgi apparatus. In the Golgi apparatus, the proteins may undergo post-translational modification before entering secretory vesicles which transport them across the cell membrane.
  • the extended cDNAs described herein have several important applications. For example, they may be used to express the entire secreted protein which they encode. Alternatively, they may be used to express portions of the secreted protein.
  • the portions may comprise the signal peptides encoded by the extended cDNAs or the mature proteins encoded by the extended cDNAs (i.e. the proteins generated when the signal peptide is cleaved off).
  • the portions may also comprise polypeptides having at least 10 consecutive amino acids encoded by the extended cDNAs. Alternatively, the portions may comprise at least 15 consecutive amino acids encoded by the extended cDNAs.
  • the portions may comprise at least 25 consecutive amino acids encoded by the extended cDNAs.
  • the portions may comprise at least 40 amino acids encoded by the extended cDNAs.
  • Antibodies which specifically recognize the entire secreted proteins encoded by the extended cDNAs or fragments thereof having at least 10 consecutive amino acids, at least 15 consecutive amino acids, at least 25 consecutive amino acids, or at least 40 consecutive amino acids may also be obtained as described below.
  • Antibodies which specifically recognize the mature protein generated when the signal peptide is cleaved may also be obtained as described below.
  • antibodies which specifically recognize the signal peptides encoded by the extended cDNAs may also be obtained.
  • the extended cDNAs may include the signal sequence.
  • the extended cDNAs may include the full coding sequence for the mature protein (i.e. the protein generated when the signal polypeptide is cleaved off).
  • the extended cDNAs may include regulatory regions upstream of the translation start site or downstream of the stop codon which control the amount, location, or developmental stage of gene expression. As discussed above, secreted proteins are therapeutically important. Thus, the proteins expressed from the cDNAs may be useful in treating or controlling a variety of human conditions.
  • the extended cDNAs may also be used to obtain the corresponding genomic DNA.
  • corresponding genomic DNA refers to the genomic DNA which encodes mRNA which includes the sequence of one of the strands of the extended cDNA in which thymidine residues in the sequence of the extended cDNA are replaced by uracil residues in the mRNA.
  • the extended cDNAs or genomic DNAs obtained therefrom may be used in forensic procedures to identify individuals or in diagnostic procedures to identify individuals having genetic diseases resulting from abnormal expression of the genes corresponding to the extended cDNAs.
  • the present DNAs are useful for constructing a high resolution map of the human chromosomes.
  • secretion vectors capable of directing the secretion of a protein of interest. Such vectors may be used in gene therapy strategies in which it is desired to produce a gene product in-one cell which is to be delivered to another location in the body. Secretion vectors may also facilitate the purification of desired proteins.
  • expression vectors capable of directing the expression of an inserted gene in a desired spatial or temporal manner or at a desired level.
  • Such vectors may include sequences upstream of the extended cDNAs such as promoters or upstream regulatory sequences.
  • the present DNAs may also be used for gene therapy to control or treat genetic diseases.
  • Signal peptides may also be fused to heterologous proteins to direct their extracellular secretion.
  • nucleic acid comprising the sequence of SEQ ID NO: 45 or a sequence complementary thereto.
  • the nucleic acid is recombinant.
  • nucleic acid comprising at least 10 consecutive bases of the sequence of SEQ ID NO: 45 or a sequence complementary thereto.
  • the nucleic acid comprises at least 15, 25, 30, 40, 50, 75, or 100 consecutive bases of the sequence of SEQ ID NO: 45 or a sequence complementary thereto.
  • the nucleic acid may be a recombinant nucleic acid.
  • nucleic acid of at least 15 bases capable of hybridizing under stringent conditions to the sequence of SEQ ID NO: 45 or a sequence complementary to the sequence of SEQ ID NO: 45.
  • the nucleic acid is recombinant.
  • nucleic acid comprising the full coding sequence of SEQ ID NO: 45, wherein the full coding sequence optionally comprises the sequence encoding signal peptide as well as the sequence encoding mature protein.
  • the nucleic acid is recombinant.
  • nucleic acid comprising the nucleotides of SEQ ID NO: 45 which encode a mature protein.
  • the nucleic acid is recombinant.
  • nucleic acid comprising the nucleotides of SEQ ID NO: 45 which encode the signal peptide.
  • the nucleic acid is recombinant.
  • nucleic acid encoding a polypeptide having the sequence of SEQ ID NO: 146.
  • nucleic acid encoding a polypeptide having the sequence of a mature protein included in the sequence of SEQ ID NO: 146.
  • nucleic acid encoding a polypeptide having the sequence of a signal peptide included in the sequence of SEQ ID NO: 146.
  • a purified or isolated polypeptide comprising at least 10 consecutive amino acids of the sequence of SEQ ID NO: 146.
  • the purified or isolated polypeptide comprises at least 15, 20, 25, 35, 50, 75, 100, 150 or 200 consecutive amino acids of the sequence of SEQ ID NO: 146.
  • the purified or isolated polypeptide comprises at least 25 consecutive amino acids of the sequence of SEQ ID NO: 146.
  • polypeptide comprising a signal peptide of the polypeptide of SEQ ID NO: 146.
  • polypeptide comprising a mature protein of the polypeptide of SEQ ID NO: 146.
  • the method further comprises the step of isolating the protein.
  • the method further comprises the step of isolating the protein.
  • a purified or isolated antibody capable of specifically binding to a protein having the sequence of SEQ ID NO: 146.
  • the antibody is capable of binding to a polypeptide comprising at least 10 consecutive amino acids of the sequence of SEQ ID NO: 146.
  • an array of cDNAs or fragments thereof of at least 15 nucleotides in length which includes at least one of the sequences of SEQ ID NOs: 40-140 and 242-377, or one of the sequences complementary to the sequences of SEQ ID NOs: 40-140 and 242-377, or a fragment thereof of at least 15 consecutive nucleotides.
  • the array includes at least two of the sequences of SEQ ID NOs: 40-140 and 242-377, the sequences complementary to the sequences of SEQ ID NOs: 40-140 and 242-377, or fragments thereof of at least 15 consecutive nucleotides.
  • the array includes at least five of the sequences of SEQ ID NOs: 40-140 and 242-377, the sequences complementary to the sequences of SEQ ID NOs: 40-140 and 242-377, or fragments thereof of at least 15 consecutive nucleotides.
  • purified polynucleotides comprising an insert from a clone deposited in a deposit having an accession number listed in Table VI or a fragment thereof comprising a contiguous span of at least 8, 10, 12, 15, 20, 25, 40, 60, 100, or 200 nucleotides of said insert.
  • polypeptides which comprise, consist of, or consist essentially of an amino acid sequence encoded by the insert from a clone deposited in a deposit having an accession number listed in Table VI, as well as polypeptides which comprise a fragment of said amino acid sequence consisting of a signal peptide, a mature protein, or a contiguous span of at least 5, 8, 10, 12, 15, 20, 25, 40, 60, 100, or 200 amino acids encoded by said insert.
  • the invention provides a purified or isolated polypeptide comprising a sequence having at least 80% identity to the polypeptide of SEQ ID NO: 146.
  • the invention provides a purified or isolated nucleic acid encoding a polypeptide comprising or consisting of SEQ ID NO: 146.
  • the invention further provides an expression vector comprising the nucleic acid and a host cell comprising the recombinant nucleic acid.
  • Also provided is a method of making the polypeptide of the invention comprising the steps of: (a) obtaining a cDNA encoding said polypeptide; (b) inserting said cDNA in an expression vector such that said cDNA is operably linked to a promoter; and (c) introducing said expression vector into a host cell whereby said host cell produces said polypeptide.
  • the invention further provides a purified or isolated antibody capable of specifically binding to the polypeptide of the invention.
  • EMBL Database Accession No. Z95114 provides a human DNA sequence from clone 212A2.
  • Duchateau et al., 1997, Journal of Biological Chemistry, 272: 25576-25582 is concerned with Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas.
  • the present extended cDNAs were obtained using 5' ESTs which were isolated as described below.
  • mRNAs having intact 5' ends must be obtained.
  • a chemical modification method involving derivatization of the 5' ends of the mRNAs and selection of the derivatized mRNAs.
  • the 5' ends of eucaryotic mRNAs possess a structure referred to as a "cap" which comprises a guanosine methylated at the 7 position.
  • the cap is joined to the first transcribed base of the mRNA by a 5', 5'-triphosphate bond.
  • the 5' guanosine is methylated in both the 2 and 7 positions.
  • the 5' guanosine is trimethylated at the 2. 7 and 7 positions.
  • the 5' cap is specifically derivatized and coupled to a reactive group on an immobilizing substrate. This specific derivatization is based on the fact that only the ribose linked to the methylated guanosine at the 5' end of the mRNA and the ribose linked to the base at the 3' terminus of the mRNA, possess 2', 3'-cis diols.
  • the 2', 3'-cis diol at the 3' end may be chemically modified, substituted, converted, or eliminated, leaving only the ribose linked to the methylated guanosine at the 5' end of the mRNA with a 2', 3'-cis diol.
  • a variety of techniques are available for eliminating the 2', 3'-cis diol on the 3' terminal ribose. For example, controlled alkaline hydrolysis may be used to generate mRNA fragments in which the 3' terminal ribose is a 3'-phosphate, 2'-phosphate or (2'. 3')-cyclophosphate.
  • RNA ligase such as T4 RNA ligase
  • RNA 1 ⁇ g was incubated in a final reaction medium of 10 ⁇ l in the presence of 5 U of T 4 phage RNA ligase in the buffer provided by the manufacturer (Gibco - BRL). 40 U of the RNase inhibitor RNasin (Promega) and, 2 ⁇ l of 32 pCp (Amersham #PB 10208).
  • the incubation was performed at 37°C for 2 hours or overnight at 7-8°C.
  • the 2', 3'-cis diol present at the 5' end of the mRNA may be oxidized using reagents such as NaBH 4 , NaBH 3 CN, or sodium periodate, thereby converting the 2', 3'-cis diol to a dialdehyde.
  • reagents such as NaBH 4 , NaBH 3 CN, or sodium periodate, thereby converting the 2', 3'-cis diol to a dialdehyde.
  • Example 2 describes the oxidation of the 2', 3'-cis diol at the 5' end of the mRNA with sodium periodate.
  • 0.1 OD unit of either a capped oligoribonucleotide of 47 nucleotides (including the cap) or an uncapped oligoribonucleotide of 46 nucleotides were treated as follows.
  • the oligoribonucleotides were produced by in vitro transcription using the transcription kit "AmpliScribe T7" (Epicentre Technologies). As indicated below, the DNA template for the RNA transcript contained a single cytosine. To synthesize the uncapped RNA, all four NTPs were included in the in vitro transcription reaction. To obtain the capped RNA, GTP was replaced by an analogue of the cap, m7G(5')ppp(5')G.
  • RNA contained a cap at its 5' end.
  • sequences of the oligoribonucleotides produced by the in vitro transcription reaction were:
  • oligoribonucleotides were dissolved in 9 ⁇ l of acetate buffer (0.1 M sodium acetate, pH 5.2) and 3 ⁇ l of freshly prepared 0.1 M sodium periodate solution. The mixture was incubated for 1 hour in the dark at 4°C or room temperature. Thereafter, the reaction was stopped by adding 4 ⁇ l of 10% ethylene glycol. The product was ethanol precipitated, resuspended in 10 ⁇ l or more of water or appropriate buffer and dialyzed against water.
  • acetate buffer 0.1 M sodium acetate, pH 5.2
  • the resulting aldehyde groups may then be coupled to molecules having a reactive amine group, such as hydrazine, carbazide, thiocarbazide or semicarbazide groups, in order to facilitate enrichment of the 5' ends of the mRNAs.
  • a reactive amine group such as hydrazine, carbazide, thiocarbazide or semicarbazide groups
  • Molecules having reactive amine groups which are suitable for use in selecting mRNAs having intact 5' ends include avidin, proteins, antibodies, vitamins, ligands capable of specifically binding to receptor molecules, or oligonucleotides.
  • Example 3 below describes the coupling of the resulting dialdehyde to biotin.
  • Example 2 The oxidation product obtained in Example 2 was dissolved in 50 ⁇ l of sodium acetate at a pH of between 5 and 5.2 and 50 ⁇ l of freshly prepared 0.02 M solution of biotin hydrazide in a methoxyethanol/water mixture (1:1) of formula:
  • n 5.
  • other commercially available hydrazides may also be used, such as molecules of the formula above in which n varies from 0 to 5.
  • the mixture was then incubated for 2 hours at 37°C. Following the incubation, the mixture was precipitated with ethanol and dialyzed against distilled water.
  • Example 4 demonstrates the specificity of the biotinylation reaction.
  • mRNAs having intact 5' ends may be enriched by binding the molecule containing a reactive amine group to a suitable solid phase substrate such as the inside of the vessel containing the mRNAs, magnetic beads, chromatography matrices, or nylon or nitrocellulose membranes.
  • a suitable solid phase substrate such as the inside of the vessel containing the mRNAs, magnetic beads, chromatography matrices, or nylon or nitrocellulose membranes.
  • the solid phase substrate may be coupled to avidin or streptavidin.
  • the molecule having the reactive amine group is an antibody or receptor ligand
  • the solid phase substrate may be coupled to the cognate antigen or receptor.
  • the molecule having a reactive amine group comprises an oligonucleotide
  • the solid phase substrate may comprise a complementary oligonucleotide.
  • the mRNAs having intact 5' ends may be released from the solid phase following the enrichment procedure.
  • the mRNAs may be released from the solid phase by simply heating to 95 degrees Celsius in 2% SOS.
  • the molecule having a reactive amine group may also be cleaved from the mRNAs having intact 5' ends following enrichment.
  • Example 5 describes the capture of biotinylated mRNAs with streptavidin coated beads and the release of the biotinylated mRNAs from the beads following enrichment.
  • the streptavidin-coated magnetic beads were prepared according to the manufacturer's instructions (CPG Inc., USA).
  • the biotinylated mRNAs were added to a hybridization buffer (1.5 M NaCl, pH 5 - 6). After incubating for 30 minutes, the unbound and nonbiotinylated material was removed. The beads were washed several times in water with 1% SDS. The beads obtained were incubated for 15 minutes at 95°C in water containing 2% SDS.
  • Example 6 demonstrates the efficiency with which biotinylated mRNAs were recovered from the streptavidin coated beads.
  • RNAs were labeled with 32 pCp, oxidized, biotinylated and bound to streptavidin coated beads as described above. Subsequently, the bound RNAs were incubated for 5, 15 or 30 minutes at 95°C in the presence of 2% SDS.
  • the products of the reaction were analyzed by electrophoresis on 12% polyacrylamide gels under denaturing conditions (7 M urea). The gels were subjected to autoradiography. During this manipulation, the hydrazone bonds were not reduced.
  • an oligonucleotide which has been derivatized to contain a reactive amine group is specifically coupled to mRNAs having an intact cap.
  • the 3' end of the mRNA is blocked prior to the step in which the aldehyde groups are joined to the derivatized oligonucleotide, as described above, so as to prevent the derivatized oligonucleotide from being joined to the 3' end of the mRNA.
  • pCp may be attached to the 3' end of the mRNA using T4 RNA ligase.
  • blocking the 3' end of the mRNA is an optional step.
  • Derivatized oligonucleotides may be prepared as described below in Example 7.
  • An oligonucleotide phosphorylated at its 3' end was converted to a 3' hydrazide in 3' by treatment with an aqueous solution of hydrazine or of dihydrazide of the formula H 2 N(R1)NH 2 at about 1 to 3 M, and at pH 4.5, in the presence of a carbodiimide type agent soluble in water such as 1-ethyl-3-(3-dimethytaminopropyl)carbodiimide at a final concentration of 0.3 M at a temperature of 8°C overnight.
  • a carbodiimide type agent soluble in water such as 1-ethyl-3-(3-dimethytaminopropyl)carbodiimide at a final concentration of 0.3 M at a temperature of 8°C overnight.
  • the derivatized oligonucleotide was then separated from the other agents and products using a standard technique for isolating oligonucleotides.
  • the mRNAs to be enriched may be treated to eliminate the 3' OH groups which may be present thereon. This may be accomplished by enzymatic ligation of sequences lacking a 3' OH, such as pCp, as described above in Example 1. Alternatively, the 3' OH groups may be eliminated by alkaline hydrolysis as described in Example 8 below.
  • the mRNAs may be treated with alkaline hydrolysis as follows. In a total volume of 100 ⁇ l of O.1N sodium hydroxide, 1.5 ⁇ g mRNA is incubated for 40 to 60 minutes at 4°C. The solution is neutralized with acetic acid and precipitated with ethanol.
  • the diol groups at the 5' ends of the mRNAs are oxidized as described below in Example 9.
  • RNA Up to 1 00 unit of RNA was dissolved in 9 ⁇ l of buffer (0.1 M sodium acetate, pH 6-7 or water) and 3 ⁇ l of freshly prepared 0.1 M sodium periodate solution. The reaction was incubated for 1 h in the dark at 4°C or room temperature. Following the incubation, the reaction was stopped by adding 4 ⁇ l of 10% ethylene glycol. Thereafter the mixture was incubated at room temperature for 15 minutes. After ethanol precipitation, the product was resuspended in 10 ⁇ l or more of water or appropriate buffer and dialyzed against water.
  • buffer 0.1 M sodium acetate, pH 6-7 or water
  • the derivatized oligonucleotide was joined to the resulting aldehydes as described in Example 10.
  • the oxidized mRNA was dissolved in an acidic medium such as 50 ⁇ l of sodium acetate pH 4-6, 50 ⁇ l of a solution of the derivatized oligonucleotide was added such that an mRNA:derivatized oligonucleotide ratio of 1:20 was obtained and mixture was reduced with a borohydride.
  • the mixture was allowed to incubate for 2 h at 37°C or overnight (14 h) at 10°C.
  • the mixture was ethanol precipitated, resuspended in 10 ⁇ l or more of water or appropriate buffer and dialyzed against distilled water. If desired, the resulting product may be analyzed using acrylamide gel electrophoresis, HPLC analysis, or other conventional techniques.
  • a reverse transcription reaction may be performed as described in Example 11 below.
  • An oligodeoxyribonucleotide was derivatized as follows. 3 00 units of an oligodeoxyribonucleotide of sequence ATCAAGAATTCGCACGAGACCATTA (SEQ 10 NO:3) having 5'-OH and 3'-P ends were dissolved in 70 ⁇ l of a 1.5 M hydroxybenzotriazole solution, pH 5.3, prepared in dimethylformamide/water (75:25) containing 2 ⁇ g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The mixture was incubated for 2 h 30 min at 22°C. The mixture was then precipitated twice in LiClO 4 /acetone.
  • the pellet was resuspended in 200 ⁇ l of 0.25 M hydrazine and incubated at 8°C from 3 to 14 h. Following the hydrazine reaction, the mixture was precipitated twice in LiClO 4 /acetone.
  • the messenger RNAs to be reverse transcribed were extracted from blocks of placenta having sides of 2 cm which had been stored at -80°C.
  • the mRNA was extracted using conventional acidic phenol techniques. Oligo-dT chromatography was used to purify the mRNAs. The integrity of the mRNAs was checked by Northern-blotting.
  • the diol groups on 7 ⁇ g of the placental mRNAs were oxidized as described above in Example 9.
  • the derivatized oligonucleotide was joined to the mRNAs as described in Example 10 above except that the precipitation step was replaced by an exclusion chromatography step to remove derivatized oligodeoxyribonucleotides which were not joined to mRNAs. Exclusion chromatography was performed as follows:
  • a glass bead (diameter 3 mm) was introduced into a 2 ml disposable pipette (length 25 cm). The pipette was filled with the gel suspension until the height of the gel stabilized at 1 cm from the top of the pipette. The column was then equilibrated with 20 ml of equilibration buffer (10 mM Tris HCl pH 7.4, 20 mM NaCl).
  • the mRNAs which had been reacted with the derivatized oligonucleotide were spotted on a nylon membrane and hybridized to a radioactive probe using conventional techniques.
  • the radioactive probe used in these hybridizations was an oligodeoxyribonucleotide of sequence TAATGGTCTCGTGCGAATTCTTGAT (SEQ ID NO:4) which was anticomplemantary to the derivatized oligonucleotide and was labeled at its 5' end with 32 P. 1/10h of the mRNAs which had been reacted with the derivatized oligonucleotide was spotted in two spots on the membrane and the membrane was visualized by autoradiography after hybridization of the probe. A signal was observed, indicating that the derivatized oligonucleotide had been joined to the mRNA.
  • the remaining 9/10 of the mRNAs which had been reacted with the derivatized oligonucleotide was reverse transcribed as follows.
  • a reverse transcription reaction was carried out with reverse transcriptase following the manufacturer's instructions.
  • 50 pmol of nonamers with random sequence were used.
  • a portion of the resulting cDNA was spotted on a positively charged nylon membrane using conventional methods.
  • the cDNAs were spotted on the membrane after the cDNA:RNA heteroduplexes had been subjected to an alkaline hydrolysis in order to eliminate the RNAs.
  • An oligonucleotide having a sequence identical to that of the derivatized oligonucleotide was labeled at its 5' end with 32 P and hybridized to the cDNA blots using conventional techniques. Single-stranded cDNAs resulting from the reverse transcription reaction were spotted on the membrane.
  • the blot contained 1 pmol, 100 fmol, 50 fmol, 10 fmol and 1 fmol respectively of a control oligodeoxyribonucleotide of sequence identical to that of the derivatized oligonucleotide.
  • the signal observed in the spots containing the cDNA indicated that approximately 15 fmol of the derivatized oligonucleotide had been reverse transcribed.
  • the single stranded cDNAs obtained after the above first strand synthesis were used as template for PCR reactions. Two types of reactions were carried out. First, specific amplification of the mRNAs for the alpha globin, dehydrogenase, pp15 and elongation factor E4 were carried out using the following pairs of oligodeoxyribonucleotide primers.
  • Non specific amplifications were also carried out with the antisense (_As) oligodeoxyribonucleotides of the pairs described above and a primer chosen from the sequence of the derivatized oligodeoxyribonucleotide (ATCAAGAATTCGCACGAGACCATTA) (SEQ ID NO:13).
  • a 1.5% agarose gel containing the following samples corresponding to the PCR products of reverse transcription was stained with ethidium bromide. (1/20th of the products of reverse transcription were used for each PCR reaction).
  • Sample 1 The products of a PCR reaction using the globin primers of SEQ ID NOs 5 and 6 in the presence of cDNA.
  • Sample 2 The products of a PCR reaction using the globin primers of SEQ ID NOs 5 and 6 in the absence of added cONA.
  • Sample 3 The products of a PCR reaction using the dehydrogenase primers of SEQ ID NOs 7 and 8 in the presence of cDNA.
  • Sample 4 The products of a PCR reaction using the dehydrogenase primers of SEQ ID NOs 7 and 8 in the absence of added cONA.
  • Sample 5 The products of a PCR reaction using the pp15 primers of SEQ ID NOs 9 and 10 in the presence of cDNA.
  • Sample 6 The products of a PCR reaction using the pp15 primers of SEQ ID NOs 9 and 10 in the absence of added cONA.
  • Sample 7 The products of a PCR reaction using the EIE4 primers of SEQ ID NOs 11 and 12 in the presence of added cDNA.
  • Sample 8 The products of a PCR reaction using the EIE4 primers of SEQ ID NOs 11 and 12 in the absence of added cDNA.
  • PCR reactions were also carried out with the antisense oligonucleotides of the globin and dehydrogenase primers (SEQ ID NOs 6 and 8) and an oligonucleotide whose sequence corresponds to that of the derivatized oligonucleotide.
  • SEQ ID NOs 6 and 8 antisense oligonucleotides of the globin and dehydrogenase primers
  • cDNAs which have been selected to include the 5' ends of the mRNAs from which they are derived.
  • the 5' ends of the mRNAs are modified as described above.
  • a reverse transcription reaction is conducted to extend a primer complementary to the mRNA to the 5' end of the mRNA.
  • Single stranded RNAs are eliminated to obtain a population of cONA/mRNA heteroduplexes in which the mRNA includes an intact 5' end.
  • the resulting heteroduplexes may be captured on a solid phase coated with a molecule capable of interacting with the molecule used to derivatize the 5' end of the mRNA.
  • Second strand cDNA synthesis may then proceed using conventional techniques. For example, the procedures disclosed in WO 96134981 or in Carninci, P. et al. High-Efficiency Full-Length cDNA Cloning by Biotinylated CAP Trapper. Genomics 37:327-336 (1996 ) may be employed to select cONAs which include the sequence derived from the 5' end of the coding sequence of the mRNA.
  • a reverse transcription reaction is conducted to extend a primer complementary to the mRNA to the 5' end of the mRNA.
  • second strand cDNA synthesis is conducted with a primer complementary to the oligonucleotide tag.
  • Figure 1 summarizes the above procedures for obtaining cDNAs which have been selected to include the 5' ends of the mRNAs from which they are derived.
  • isolated mRNA is treated with alkaline phosphatase to remove the phosphate groups present on the 5' ends of uncapped incomplete mRNAs.
  • a decapping enzyme such as T4 polynucleotide kinase or tobacco acid pyrophosphatase.
  • An oligonucleotide which may be either a DNA oligonucleotide or a DNA-RNA hybrid oligonucleotide having RNA at its 3' end, is then ligated to the phosphate present at the 5' end of the decapped mRNA using T4 RNA ligase.
  • the oligonucleotide may include a restriction site to facilitate cloning of the cONAs following their synthesis.
  • Example 12 below describes one enzymatic method based on the doctoral thesis of Dumas.
  • RNA was dephosphorylated using Calf Intestinal Phosphatase (Biolabs). After a phenol chloroform extraction, the cap structure of mRNA was hydrolysed using the Tobacco Acid Pyrophosphatase (purified as described by Shinshi et al., Biochemistry 15: 2185-2190, 1976 ) and a hemi 5'DNA/RNA-3' oligonucleotide having an unphosphorylated 5' end, a stretch of adenosine ribophosphate at the 3' end, and an EcoRI site near the 5' end was ligated to the 5'P ends of mRNA using the T4 RNA ligase (Biolabs).
  • Tobacco Acid Pyrophosphatase purified as described by Shinshi et al., Biochemistry 15: 2185-2190, 1976
  • a hemi 5'DNA/RNA-3' oligonucleotide having an unphosphorylated 5' end, a stretch of adenosine
  • Oligonucleotides suitable for use in this procedure are preferably 30-50 bases in length. Oligonucleotides having an unphosphorylated 5' end may be synthesized by adding a fluorochrome at the 5' end. The inclusion of a stretch of adenosine ribophosphates at the 3' end of the oligonucleotide increases ligation efficiency. It will be appreciated that the oligonucleotide may contain cloning sites other than EcoRI.
  • first and second strand cDNA synthesis may be carried out using conventional methods or those specified in EPO 625,572 and Kato et al. Construction of a Human Full-Length cONA Bank. Gene 150:243-250 (1994 ), and Dumas Milne Edwards, supra .
  • the resulting cDNA may then be ligated into vectors such as those disclosed in Kato et al. Construction of a Human Full-Length cDNA Bank. Gene 150:243-250 (1994 ) or other nucleic acid vectors known to those skilled in the art using techniques such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2d Ed., Cold Spring Harbor Laboratory Press, 1989 .
  • mRNAs were prepared as described in Example 13 below.
  • RNAs or PolyA+ RNAs derived from 29 different tissues were respectively purchased from LABIMO and CLONTECH and used to generate 44 cDNA libraries as described below. The purchased RNA had been isolated from cells or tissues using acid guanidium thiocyanate-phenol-chloroform extraction ( Chomczyniski, P and Sacchi, N., Analytical Biochemistry 162:156-159, 1987 ). PolyA+ RNA was isolated from total RNA (LABIMO) by two passes of oligodT chromatography, as described by Aviv and Leder ( Aviv, H. and Leder, P., Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972 ) in order to eliminate ribosomal RNA.
  • RNAs blots a probe derived from the sequence of the 28S RNA.
  • Preparations of mRNAs with less than 5% of ribosomal RNAs were used in library construction. To avoid constructing libraries with RNAs contaminated by exogenous sequences (prokaryotic or fungal), the presence of bacterial 16S ribosomal sequences or of two highly expressed mRNAs was examined using PCR.
  • oligonucleotide tag was attached to the cap at the 5' ends of the mRNAs.
  • the oligonucleotide tag had an EcoRI site therein to facilitate later cloning procedures.
  • the integrity of the mRNA was examined by performing a Northern blot with 200-500ng of mRNA using a probe complementary to the oligonucleotide tag.
  • first strand cDNA synthesis was performed using reverse transcriptase with random nonamers as primers.
  • methylated dCTP was used for first strand synthesis.
  • the first strand of cONA was precipitated using isopropanol in order to eliminate residual primers.
  • the second strand of the cDNA was synthesized with a Klenow fragment using a primer corresponding to the 5'end of the ligated oligonucleotide described in Example 12.
  • the primer is 20-25 bases in length.
  • Methylated dCTP was also used for second strand synthesis in order to protect internal EcoRI sites in the cDNA from digestion during the cloning process.
  • the ends of the cDNA were blunted with T4 DNA polymerase (Biolabs) and the cDNA was digested with EcoRI. Since methylated dCTP was used during cDNA synthesis, the EcoRI site present in the tag was the only site which was hemi-methylated. Consequently, only the EcoRI site in the oligonucleotide tag was susceptible to EcoRI digestion.
  • the cONA was then size fractionated using exclusion chromatography (AcA, Biosepra). Fractions corresponding to cDNAs of more than 150 bp were pooled and ethanol precipitated.
  • the cDNA was directionally cloned into the Smal and EcoRI ends of the phagemid pBlueScript vector (Stratagene). The ligation mixture was electroporated into bacteria and propagated under appropriate antibiotic selection. Clones containing the oligonucleotide tag attached were selected as described in Example 16 below.
  • the plasmid ONAs containing 5' EST libraries made as described above were purified (Qiagen).
  • a positive selection of the tagged clones was performed as follows. Briefly, in this selection procedure, the plasmid DNA was converted to single stranded DNA using gene II endonuclease of the phage F1 in combination with an exonuclease ( Chang et al., Gene 127:95-8, 1993 ) such as exonuclease III or T7 gene 6 exonuclease.
  • the resulting single stranded DNA was then purified using paramagnetic beads as described by Fry et al, Biotechniques, 13: 124-131, 1992 .
  • the single stranded DNA was hybridized with a biotinylated oligonucleotide having a sequence corresponding to the 3' end of the oligonucleotide described in Example 13.
  • the primer has a length of 20-25 bases.
  • Clones including a sequence complementary to the biotinylated oligonucleotide were captured by incubation with streptavidin coated magnetic beads followed by magnetic selection. After capture of the positive clones, the plasmid DNA was released from the magnetic beads and converted into double stranded DNA using a DNA polymerase such as the ThermoSequenase obtained from Amersham Pharmacia Biotech.
  • protocols such as the Gene Trapper kit (Gibco BRL) may be used.
  • the double stranded DNA was then electroporated into bacteria.
  • the percentage of positive clones having the 5' tag oligonucleotide was estimated to typically rank between 90 and 98% using dot blot analysis.
  • MTP microtiter plates
  • Plasmid inserts were first amplified by PCR on PE 9600 thermocyclers (Perkin-Elmer), using standard SETA-A and SETA-B primers (Genset SA), AmpliTaqGold (Perkin-Elmer), dNTPs (Boehringer), buffer and cycling conditions as recommended by the Perkin-Elmer Corporation.
  • PCR products were then sequenced using automatic ABI Prism 377 sequencers (Perkin Elmer, Applied Biosystems Division, Foster City, CA). Sequencing reactions were performed using PE 9600 thermocyclers (Perkin Elmer) with standard dye-primer chemistry and ThermoSequenase (Amersham Life Science). The primers used were either T7 or 21M13 (available from Genset SA) as appropriate. The primers were labeled with the JOE, FAM, ROX and TAMRA dyes. The dNTPs and ddNTPs used in the sequencing reactions were purchased from Boehringer. Sequencing buffer, reagent concentrations and cycling conditions were as recommended by Amersham.
  • sequence data from the 44 cDNA libraries made as described above were transferred to a proprietary database, where quality control and validation steps were performed.
  • a proprietary base-caller (“Trace"), working using a Unix system automatically flagged suspect peaks, taking into account the shape of the peaks, the inter-peak resolution, and the noise level.
  • the proprietary base-caller also performed an automatic trimming. Any stretch of 25 or fewer bases having more than 4 suspect peaks was considered unreliable and was discarded.
  • Sequences corresponding to cloning vector or ligation oligonucleotides were automatically removed from the EST sequences. However, the resulting EST sequences may contain 1 to 5 bases belonging to the above mentioned sequences at their 5' end. If needed, these can easily be removed on a case by case basis.
  • Computer readable media include magnetically readable media, optically readable media, or electronically readable media.
  • the computer readable media may be a hard disc, a floppy disc, a magnetic tape, CD-ROM, RAM, or ROM as well as other types of other media known to those skilled in the art.
  • sequence data may be stored and manipulated in a variety of data processor programs in a variety of formats.
  • sequence data may be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE.
  • the computer readable media on which the sequence information is stored may be in a personal computer, a network, a server or other computer systems known to those skilled in the art.
  • the computer or other system preferably includes the storage media described above, and a processor for accessing and manipulating the sequence data.
  • sequence data may be manipulated and searched to locate those stored sequences which contain a desired nucleic acid sequence or which encode a protein having a particular functional domain.
  • the stored sequence information may be compared to other known sequences to identify homologies, motifs implicated in biological function, or structural motifs.
  • Programs which may be used to search or compare the stored sequences include the MacPattern (EMBL), BLAST, and BLAST2 program series (NCBI), basic local alignment search tool programs for nucleotide (BLASTN) and peptide (BLASTX) comparisons ( Altschul et al, J. Mol. Biol. 215: 403 (1990 )) and FASTA ( Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988 )).
  • the BLAST programs then extend the alignments on the basis of defined match and mismatch criteria.
  • Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
  • ESTs in the NETGENETM database which were derived from undesired sequences such as transfer RNAs, ribosomal RNAs, mitochondrial RNAs, procaryotic RNAs, fungal RNAs, Alu sequences, LI sequences, or repeat sequences were identified using the FASTA and BLASTN programs with the parameters listed in Table II.
  • the 5' EST sequences were compared to the sequences of 1190 known tRNAs obtained from EMBL release 38, of which 100 were human. The comparison was performed using FASTA on both strands of the 5' ESTs. Sequences having more than 80% homology over more than 60 nucleotides were identified as tRNA. Of the 144,341 sequences screened, 26 were identified as tRNAs and eliminated from further consideration.
  • the 5' EST sequences were compared to the sequences of 2497 known rRNAs obtained from EMBL release 38, of which 73 were human. The comparison was performed using BLASTN on both strands of the 5' ESTs with the parameter S-108. Sequences having more than 80% homology over stretches longer than 40 nucleotides were identified as rRNAs. Of the 144,341 sequences screened, 3,312 were identified as rRNAs and eliminated from further consideration.
  • Sequences which might have resulted from exogenous contaminants were eliminated from further consideration by comparing the 5' EST sequences to release 46 of the EMBL bacterial and fungal divisions using BLASTN with the parameter S-144. All sequences having more than 90% homology over at least 40 nucleotides were identified as exogenous contaminants. Of the 42 cDNA libraries examined, the average percentages of procaryotic and fungal sequences contained therein were 0.2% and 0.5% respectively. Among these sequences, only one could be identified as a sequence specific to fungi. The others were either fungal or procaryotic sequences having homologies with vertebrate sequences or including repeat sequences which had not been masked during the electronic comparison.
  • the 5' ESTs were compared to 6093 Alu sequences and 1115 L1 sequences to mask 5' ESTs containing such repeat sequences from further consideration.
  • 5' ESTs including THE and MER repeats, SSTR sequences or satellite, micro-satellite, or telomeric repeats were also eliminated from further consideration.
  • 11.5% of the sequences in the libraries contained repeat sequences. Of this 11.5%, 7% contained Alu repeals, 3.3% contained L1 repeats and the remaining 1.2% were derived from the other types of repetitive sequences which were screened.
  • These percentages are consistent with those found in cDNA libraries prepared by other groups.
  • the cDNA libraries of Adams et al. contained between 0% and 7.4% Alu repeats depending on the source of the RNA which was used to prepare the cDNA library ( Adams et al., Nature 377:174, 1996 ).
  • sequences of those 5' ESTs remaining after the elimination of undesirable sequences were compared with the sequences of known human mRNAs to determine the accuracy of the sequencing procedures described above.
  • sequences of 5' ESTs derived from known sequences were identified and compared to the known sequences.
  • a FASTA analysis with overhangs shorter than 5 bp on both ends was conducted on the 5' ESTs to identify those matching an entry in the public human mRNA database.
  • the 6655 5' ESTs which matched a known human mRNA were then realigned with their cognate mRNA and dynamic programming was used to include substitutions, insertions, and deletions in the list of "errors" which would be recognized. Errors occurring in the last 10 bases of the 5' EST sequences were ignored to avoid the inclusion of spurious cloning sites in the analysis of sequencing accuracy.
  • the sequences of the ends of the 5' ESTs which were derived from the elongation factor 1 subunit a and ferritin heavy chain genes were compared to the known cDNA sequences for these genes. Since the transcription start sites for the elongation factor 1 subunit ⁇ and ferritin heavy chain are well characterized, they may be used to determine the percentage of 5' ESTs derived from these genes which included the authentic transcription start sites.
  • cDNAs included sequences close to or upstream of the 5' end of the corresponding mRNAs.
  • the EST libraries made above included multiple 5' ESTs derived from the same mRNA. The sequences of such 5' ESTs were compared to one another and the longest 5' ESTs for each mRNA were identified. Overlapping cDNAs were assembled into continuous sequences (contigs). The resulting continuous sequences were then compared to public databases to gauge their similarity to known sequences, as described in Example 21 below.
  • HSPs High Scoring Segment Pairs
  • NR 100 X (Number of new unique sequences found in the library/Total number of sequences from the library). Typically, novelty rating range between 10% and 41 % depending on the tissue from which the EST library was obtained. For most of the libraries, the random sequencing of 5' EST libraries was pursued until the novelty rate reached 20%.
  • the 5' ESTs in the NETGENETM database were screened to identify those having an uninterrupted open reading frame (ORF) longer than 45 nucleotides beginning with an ATG codon and extending to the end of the EST. Approximately half of the cDNA sequences in NETGENETM contained such an ORF. The ORFs of these 5' ESTs were searched to identify potential signal motifs using slight modifications of the procedures disclosed in Von Heijne, G. A New Method for Predicting Signal Sequence Cleavage Sites. Nucleic Acids Res. 14:4683-4690 (1986 ).
  • Example 23 To confirm the accuracy of the above method for identifying signal sequences, the analysis of Example 23 was performed.
  • the accuracy of the above procedure for identifying signal sequences encoding signal peptides was evaluated by applying the method to the 43 amino terminal amino acids of all human SwissProt proteins.
  • the computed Von Heijne score for each protein was compared with the known characterization of the protein as being a secreted protein or a non-secreted protein. In this manner, the number of non-secreted proteins having a score higher than 3.5 (false positives) and the number of secreted proteins having a score lower than 3.5 (false negatives) could be calculated.
  • the signal sequences from the 5' ESTs may be cloned into a vector designed for the identification of signal peptides.
  • Some signal peptide identification vectors are designed to confer the ability to grow in selective medium on host cells which have a signal sequence operably inserted into the vector.
  • the signal sequence of the 5' EST may be inserted upstream and in frame with a non secreted form of the yeast invertase gene in signal peptide selection vectors such as those described in U.S. Patent No. 5,536,637 . Growth of host cells containing signal sequence selection vectors having the signal sequence from the 5' EST inserted therein confirms that the 5' EST encodes a genuine signal peptide.
  • the presence of a signal peptide may be confirmed by cloning the extended cDNAs obtained using the ESTs into expression vectors such as pXT1 (as described below), or by constructing promoter-signal sequence reporter gene vectors which encode fusion proteins between the signal peptide and an assayable reporter protein.
  • expression vectors such as pXT1 (as described below)
  • promoter-signal sequence reporter gene vectors which encode fusion proteins between the signal peptide and an assayable reporter protein.
  • the growth medium may be harvested and analyzed for the presence of the secreted protein. The medium from these cells is compared to the medium from cells containing vectors lacking the signal sequence or extended cDNA insert to identify vectors which encode a functional signal peptide or an authentic secreted protein.
  • EST-ext Those 5' ESTs having a sequence not matching any vertebrate sequence but matching a publicly known EST were designated "EST-ext", provided that the known EST sequence was extended by at least 40 nucleotides in the 5' direction.
  • EST-ext Those 5' ESTs having a Von Heijne's score of at least 3.5 fell into this category.
  • ESTs not matching any vertebrate sequence but matching a publicly known EST without extending the known EST by at least 40 nucleotides in the 5' direction were designated "EST.”
  • sequences in the SIGNALTAGTM database 599 of the 5' ESTs having a Von Heijne's score of at least 3.5 fell into this category.
  • Figure 4 shows the distribution of 5' ESTs in each category and the number of 5' ESTs in each category having a given minimum von Heijne's score.
  • Each of the 5' ESTs was categorized based on the tissue from which its corresponding mRNA was obtained, as described below in Example 25.
  • Figure 5 shows the tissues from which the mRNAs corresponding to the 5' ESTs in each of the above described categories were obtained.
  • the spatial and temporal expression patterns of the mRNAs corresponding to the 5' ESTs, as well as their expression levels may be determined as described in Example 26 below. Characterization of the spatial and temporal expression patterns and expression levels of these mRNAs is useful for constructing expression vectors capable of producing a desired level of gene product in a desired spatial or temporal manner, as will be discussed in more detail below.
  • 5' ESTs whose corresponding mRNAs are associated with disease states may also be identified.
  • a particular disease may result from lack of expression, over expression, or under expression of an mRNA corresponding to a 5' EST.
  • 5' ESTs responsible for the disease may be identified.
  • Expression levels and patterns of mRNAs corresponding to 5' ESTs or extended cDNAs may be analyzed by solution hybridization with long probes as described in International Patent Application No. WO 97/05277 . Briefly, a 5' EST, extended cDNA, or fragment thereof corresponding to the gene encoding the mRNA to be characterized is inserted at a cloning site immediately downstream of a bacteriophage (T3, T7 or SP6) RNA polymerase promoter to produce antisense RNA.
  • the 5' EST or extended cDNA has 100 or more nucleotides.
  • the plasmid is linearized and transcribed in the presence of ribonucleotides comprising modified ribonucleotides (i.e. biotin-UTP and DIG-UTP).
  • ribonucleotides comprising modified ribonucleotides (i.e. biotin-UTP and DIG-UTP).
  • An excess of this doubly labeled RNA is hybridized in solution with mRNA isolated from cells or tissues of interest.
  • the hybridizations are performed under standard stringent conditions (40-50°C for 16 hours in an 80% formamide, 0.4 M NaCl buffer, pH 7-8).
  • the unhybridized probe is removed by digestion with ribonucleases specific for single-stranded RNA (i.e. RNases CL3, T1, Phy M, U2 or A).
  • the presence of the biotin-UTP modification enables capture of the hybrid on a microtitration plate coated with streptavidin.
  • the presence of the DIG modification enables the hybrid to be detected and quantified by ELISA using an anti-DIG antibody coupled to alkaline phosphatase.
  • the 5' ESTs, extended cDNAs, or fragments thereof may also be tagged with nucleotide sequences for the serial analysis of gene expression (SAGE) as disclosed in UK Patent Application No. 2 305 241 A .
  • SAGE serial analysis of gene expression
  • cDNAs are prepared from a cell, tissue, organism or other source of nucleic acid for which it is desired to determine gene expression patterns.
  • the resulting cDNAs are separated into two pools.
  • the cDNAs in each pool are cleaved with a first restriction endonuclease, called an "anchoring enzyme," having a recognition site which is likely to be present at least once in most cDNAs.
  • the fragments which contain the 5' or 3' most region of the cleaved cDNA are isolated by binding to a capture medium such as streptavidin coated beads.
  • a first oligonucleotide linker having a first sequence for hybridization of an amplification primer and an internal restriction site for a "tagging endonuclease" is ligated to the digested cDNAs in the first pool. Digestion with the second endonuclease produces short "tag" fragments from the cDNAs.
  • a second oligonucleotide having a second sequence for hybridization of an amplification primer and an internal restriction site is ligated to the digested cDNAs in the second pool.
  • the cDNA fragments in the second pool are also digested with the "tagging endonuclease" to generate short "tag” fragments derived from the cDNAs in the second pool.
  • the "tags" resulting from digestion of the first and second pools with the anchoring enzyme and the tagging endonuclease are ligated to one another to produce "ditags.”
  • the ditags are concatamerized to produce ligation products containing from 2 to 200 ditags.
  • the tag sequences are then determined and compared to the sequences of the 5' ESTs or extended cDNAs to determine which 5' ESTs or extended cDNAs are expressed in the cell, tissue, organism, or other source of nucleic acids from which the tags were derived. In this way, the expression pattern of the 5' ESTs or extended cDNAs in the cell, tissue, organism, or other source of nucleic acids is obtained.
  • arrays means a one dimensional, two dimensional, or multidimensional arrangement of full length cDNAs (i.e. extended cDNAs which include the coding sequence for the signal peptide, the coding sequence for the mature protein, and a stop codon), extended cDNAs, 5' ESTs or fragments of the full length cDNAs, extended cDNAs, or 5' ESTs of sufficient length to permit specific detection of gene expression.
  • the fragments are at least 15 nucleotides in length. More preferably, the fragments are at least 100 nucleotides in length. More preferably, the fragments are more than 100 nucleotides in length. In some embodiments the fragments may be more than 500 nucleotides in length.
  • cDNAs full length cDNAs, extended cDNAs, 5' ESTs, or fragments thereof in a complementary DNA microarray as described by Schena et al. ( Science 270 :467-470, 1995; Proc. Natl. Acad. Sci. U.S.A . 93 :10614-10619, 1996).
  • Full length cDNAs, extended cDNAs, 5' ESTs or fragments thereof are amplified by PCR and arrayed from 96-well microtiter plates onto silylated microscope slides using high-speed robotics.
  • Printed arrays are incubated in a humid chamber to allow rehydration of the array elements and rinsed, once in 0.2% SDS for 1 min, twice in water for 1 min and once for 5 min in sodium borohydride solution.
  • the arrays are submerged in water for 2 min at 95°C, transferred into 0.2% SDS for 1 min, rinsed twice with water, air dried and stored in the dark at 25°C.
  • Probes are hybridized to 1 cm 2 microarrays under a 14 x 14 mm glass coverslip for 6-12 hours at 60°C. Arrays are washed for 5 min at 25°C in low stringency wash buffer (1 x x SSC/0.2% SDS), then for 10 min at room temperature in high stringency wash buffer (0.1 x SSC/0.2% SDS). Arrays are scanned in 0.1 x SSC using a fluorescence laser scanning device fitted with a custom filter set. Accurate differential expression measurements are obtained by taking the average of the ratios of two independent hybridizations.
  • Quantitative analysis of the expression of genes may also be performed with full length cDNAs, extended cDNAs, 5' ESTs, or fragments thereof in complementary DNA arrays as described by Pietu et al. (Genome Research 6:492-503, 1996 ).
  • the full length cDNAs, extended cDNAs, 5' ESTs or fragments thereof are PCR amplified and spotted on membranes.
  • mRNAs originating from various tissues or cells are labeled with radioactive nucleotides. After hybridization and washing in controlled conditions, the hybridized mRNAs are detected by phospho-imaging or autoradiography. Duplicate experiments are performed and a quantitative analysis of differentially expressed mRNAs is then performed.
  • expression analysis of the 5' ESTs or extended cDNAs can be done through high density nucleotide arrays as described by Lockhart et al. (Nature Biotechnology 14: 1675-1680, 1996 ) and Sosnowsky et al (Proc. Natl. Acad. Sci. 94:1119-1123, 19971 .
  • Oligonucleotides of 15-50 nucleotides corresponding to sequences of the 5' ESTs or extended cDNAs are synthesized directly on the chip (Lockhart et al.. supra ) or synthesized and then addressed to the chip (Sosnowski et al., supra ).
  • the oligonucleotides are about 20 nucleotides in length.
  • cDNA probes labeled with an appropriate compound such as biotin, digoxigenin or fluorescent dye, are synthesized from the appropriate mRNA population and then randomly fragmented to an average size of 50 to 100 nucleotides. The said probes are then hybridized to the chip. After washing as described in Lockhart et al., supra and application of different electric fields ( Sosnowsky et al., Proc. Natl. Acad. Sci. 94:119-1123 ), the dyes or labeling compounds are detected and quantified. Duplicate hybridizations are performed.
  • an appropriate compound such as biotin, digoxigenin or fluorescent dye
  • Comparative analysis of the intensity of the signal originating from cDNA probes on the same target oligonucleotide in different cDNA samples indicates a differential expression of the mRNA corresponding to the 5' EST or extended cDNA from which the oligonucleotide sequence has been designed.
  • the extended cDNAs may include the entire coding sequence of the protein encoded by the corresponding mRNA, including the authentic translation start site, the signal sequence, and the sequence encoding the mature protein remaining after cleavage of the signal peptide. Such extended cDNAs are referred to herein as "full length cDNAs.” Alternatively, the extended cDNAs may include only the sequence encoding the mature protein remaining after cleavage of the signal peptide, or only the sequence encoding the signal peptide.
  • Example 27 describes a general method for obtaining extended cDNAs.
  • Example 28 describes the cloning and sequencing of several extended cDNAs, including extended cDNAs which include the entire coding sequence and authentic 5' end of the corresponding mRNA for several secreted proteins.
  • Examples 27, 28, and 29 can also be used to obtain extended cDNAs which encode less than the entire coding sequence of the secreted proteins encoded by the genes corresponding to the 5' ESTs.
  • the extended cDNAs isolated using these methods encode at least 10 amino acids of the protein encoded by the sequence of SEO ID NO: 45.
  • the extended cDNAs encode at least 20 amino acids of the protein encoded by the sequence of SEO ID NOs: 45.
  • the extended cDNAs encode at least 30 amino amino acids of the sequence of SEQ ID NOs: 45
  • the extended cDNAs encode a full length protein sequence, which includes the protein coding sequence of SEQ ID NO: 45.
  • the method takes advantage of the known 5' sequence of the mRNA.
  • a reverse transcription reaction is conducted on purified mRNA with a poly 14dT primer containing a 49 nucleotide sequence at its 5' end allowing the addition of a known sequence at the end of the cDNA which corresponds to the 3' end of the mRNA.
  • the primer may have the following sequence: 5'-ATC GTT GAG ACT CGT ACC AGC AGA GTC ACG AGA GAG ACT ACA CGG TAC TGG TTT TTT TTT TTT TTVN -3' (SEQ ID NO:14).
  • sequences may also be added to the poly dT sequence and used to prime the first strand synthesis.
  • RNAs Using this primer and a reverse transcriptase such as the Superscript II (Gibco BRL) or Rnase H Minus M-MLV (Promega) enzyme, a reverse transcript anchored at the 3' polyA site of the RNAs is generated.
  • a reverse transcriptase such as the Superscript II (Gibco BRL) or Rnase H Minus M-MLV (Promega) enzyme
  • a pair of nested primers on each end is designed based on the known 5' sequence from the 5' EST and the known 3' end added by the poly dT primer used in the first strand synthesis.
  • Software used to design primers are either based on GC content and melting temperatures of oligonucleotides, such as OSP ( Illier and Green, PCA Meth. Appl. 1:124.128. 1991 ), or based on the octamer frequency disparity method ( Griffais et al., Nucleic Acids Res. 19: 3887-3891, 1991 such as PC-Rare (http://bioinformatics.weizmann.ac.il/soflware/PC.Rare/doc/manuel.html).
  • the nested primers at the 5' end are separated from one another by four to nine bases.
  • the 5' primer sequences may be selected to have melting temperatures and specificities suitable for use in PCR.
  • the nested primers at the 3' end are separated from one another by four to nine bases.
  • the nested 3' primers may have the following sequences: (5'- CCA GCA GAG TCA CGA GAG AGA CTA CAC GG -3'(SEQ ID NO:15), and 5'- CAC GAG AGA GAC TAC ACG GTA CTG G -3' (SEO ID NO:16). These primers were selected because they have melting temperatures and specificities compatible with their use in PCR. However, those skilled in the art will appreciate that other sequences may also be used as primers.
  • the first PCR run of 25 cycles is performed using the Advantage Tth Polymerase Mix (Clontech) and the outer primer from each of the nested pairs.
  • a second 20 cycle PCR using the same enzyme and the inner primer from each of the nested pairs is then performed on 1/2500 of the first PCR product. Thereafter, the primers and nucleotides are removed.
  • the second 5' primer is located upstream of the translation initiation codon thus yielding a nested PCR product containing the whole coding sequence.
  • a full length extended cDNA undergoes a direct cloning procedure as described in section a below.
  • the second 5' primer is located downstream of the translation initiation codon, thereby yielding a PCR product containing only part of the ORF.
  • incomplete PCR products are submitted to a modified procedure described in section b below.
  • the resulting nested PCR product contains the complete coding sequence, as predicted from the 5'EST sequence, it is cloned in an appropriate vector such as pED6dpc2, as described in section 3.
  • the complete coding sequence can be assembled from several partial sequences determined directly from different PCR products as described in the following section.
  • Sequencing of extended cDNAs is performed using a Die Terminator approach with the AmpliTaq DNA polymerase FS kit available from Perkin Elmer.
  • primer walking is performed using software such as OSP to choose primers and automated computer software such as ASMG ( Sutton et al., Genome Science Technol. 1: 9.19, 1995 ) to construct contigs of walking sequences including the initial 5' tag using minimum overlaps of 32 nucleotides.
  • ASMG Sutton et al., Genome Science Technol. 1: 9.19, 1995
  • primer walking is performed until the sequences of full length cDNAs are obtained.
  • the length of the sequence determined from the computer analysis is not at least 70% of the length of the nested PCR product, these PCR products are cloned and the sequence of the insertion is determined.
  • the size of the mRNA detected for a given PCR product is used to finally assess that the sequence is complete. Sequences which do not fulfill the above criteria are discarded and will undergo a new isolation procedure.
  • Sequence data of all extended cDNAs are then transferred to a proprietary database, where quality controls and validation steps are carried out as described in example 15.
  • the PCR product containing the full coding sequence is then cloned in an appropriate vector.
  • the extended cDNAs can be cloned into the expression vector pED6dpc2 (DiscoverEase, Genetics Institute, Cambridge, MA) as follows.
  • the structure of pED6dpc2 is shown in Figure 7 .
  • pED6dpc2 vector DNA is prepared with blunt ends by performing an EcoRI digestion followed by a fill in reaction.
  • the blunt ended vector is dephosphorylated.
  • the PCR product containing the full coding sequence or the extended cDNA obtained as described above is phosphorylated with a kinase subsequently removed by phenol-Sevag extraction and precipitation.
  • the double stranded extended cDNA is then ligated to the vector and the resulting expression plasmid introduced into appropriate host cells.
  • the orientation of several clones for each PCR product is determined. Then, 4 to 10 clones are ordered in microtiter plates and subjected to a PCR reaction using a first primer located in the vector close to the cloning site and a second primer located in the portion of the extended cDNA corresponding to the 3' end of the mRNA.
  • This second primer may be the antisense primer used in anchored PCR in the case of direct cloning (case a) or the antisense primer located inside the 3'UTR in the case of indirect cloning (case b).
  • Clones in which the start codon of the extended cDNA is operably linked to the promoter in the vector so as to permit expression of the protein encoded by the extended cDNA are conserved and sequenced.
  • approximately 50 bp of vector DNA on each side of the cDNA insert are also sequenced.
  • the cloned PCR products are then entirely sequenced according to the aforementioned procedure.
  • contig assembly of long fragments is then performed on walking sequences that have already contigated for uncloned PCR products during primer walking.
  • Sequencing of cloned amplicons is complete when the resulting contigs include the whole coding region as well as overlapping sequences with vector DNA on both ends.
  • extended cDNAs are compared to the known sequences of vector RNA using the FASTA program. Sequences of extended cDNAs with more than 90% homology over stretches of 15 nucleotides are identified as vector RNA.
  • extended cDNA sequences were compared to the sequences of 1190 known tRNAs obtained from EMBL release 38, of which 100 were human. Sequences of extended cDNAs having more than 80% homology over 60 nucleotides using FASTA were identified as tRNA.
  • extended cDNA sequences were compared to the sequences of 2497 known rRNAs obtained from EMBL release 38, of which 73 were human. Sequences of extended cDNAs having more than 80% homology over stretches longer than 40 nucleotides using BLASTN were identified as rRNAs..
  • extended cDNA sequences were compared to the sequences of the two known mitochondrial genomes for which the entire genomic sequences are available and all sequences transcribed from these mitochondrial genomes including tRNAs, rRNAs, and mRNAs for a total of 38 sequences. Sequences of extended cDNAs having more than 80% homology over stretches longer than 40 nucleotides using BLASTN were identified as mtRNAs.
  • Sequences which might have resulted from other exogenous contaminants were identified by comparing extended cDNA sequences to release 105 of Genbank bacterial and fungal divisions. Sequences of extended cDNAs having more than 90% homology over 40 nucleotides using BLASTN were identified as exogenous prokaryotic or fungal contaminants.
  • extended cDNAs were searched for different repeat sequences, including Alu sequences, L1 sequences, THE and MER repeats, SSTR sequences or satellite, micro-satellite, or telomeric repeats. Sequences of extended cDNAs with more than 70% homology over 40 nucleotide stretches using BLASTN were identified as repeat sequences and masked in further identification procedures. In addition, clones showing extensive homology to repeats, i.e., matches of either more than 50 nucleotides if the homology was at least 75% or more than 40 nucleotides if the homology was at least 85% or more than 30 nucleotides if the homology was at least 90%, were flagged.
  • Structural features e.g. polyA tail and polyadenylation signal, of the sequences of full length extended cDNAs are subsequently determined as follows.
  • a polyA tail is defined as a homopolymeric stretch of at least 11 A with at most one alternative base within it.
  • the polyA tail search is restricted to the last 20 nt of the sequence and limited to stretches of 11 consecutive A's because sequencing reactions are often not readable after such a polyA stretch. Stretches with 100% homology over 6 nucleotides are identified as polyA tails.
  • the polyA tail is clipped from the full-length sequence.
  • the 50 bp preceding the polyA tail are searched for the canonic polyadenylation AAUAAA signal allowing one mismatch to account for possible sequencing errors and known variation in the canonical sequence of the polyadenylation signal.
  • the 3 upper strand frames of extended cDNAs are searched for ORFs defined as the maximum length fragments beginning with a translation initiation codon and ending with a stop codon. ORFs encoding at least 20 amino acids are preferred.
  • Each found ORF is then scanned for the presence of a signal peptide in the first 50 amino-acids or, where appropriate, within shorter regions down to 20 amino acids or less in the ORF, using the matrix method of von Heijne (Nuc. Acids Res. 14: 4683-4690 (1986 )) and the modification described in Example 22.
  • Sequences of full length extended cDNAs are then compared to known sequences on a nucleotidic or proteic basis.
  • Sequences of full length extended cDNAs are compared to the following known nucleic acid sequences: vertebrate sequences (Genbank), EST sequences (Genbank), patented sequences (Geneseqn) and recently identified sequences (Genbank daily releases) available at the time of filing for the priority documents.
  • Full length cDNA sequences are also compared to the sequences of a private database (Genset internal sequences) in order to find sequences that have already been identified by applicants.
  • Sequences of full length extended cONAs with more than 90% homology over 30 nucleotides using either BLASTN or BLAST2N as indicated in Table III are identified as sequences that have already been described. Matching vertebrate sequences are subsequently examined using FASTA; full length extended cONAs with more than 70% homology over 30 nucleotides are identified as sequences that have already been described.
  • ORFs encoded by full length extended cDNAs as defined in section c) are subsequently compared to known amino acid sequences found in Swissprot release CHP, PIR release PIR# and Genpept release GPEPT public databases using BLASTP with the parameter W-8 and allowing a maximum of 10 matches. Sequences of full length extended cDNAs showing extensive homology to known protein sequences are recognized as already identified proteins.
  • ORFs containing a signal peptide and ending either before the polyA tail (case a) or before the end of the cloned 3'UTR (case b) are kept. Then, ORFs containing unlikely mature proteins such as mature proteins which size is less than 20 amino acids or less than 25% of the immature protein size are eliminated.
  • Sequences of full length extended cDNA clones are then compared pairwise with BLAST after masking of the repeat sequences. Sequences containing at least 90% homology over 30 nucleotides are clustered in the same class. Each cluster is then subjected to a cluster analysis that detects sequences resulting from internal priming or from alternative splicing, identical sequences or sequences with several frameshifts. This automatic analysis serves as a basis for manual selection of the sequences.
  • Selection of full length extended cDNA clones encoding sequences of interest is performed using the following criteria. Structural parameters (initial tag, polyadenylation site and signal) are first checked. Then, homologies with known nucleic acids and proteins are examined in order to determine whether the clone sequence match a known nucleic/proteic sequence and, in the latter case, its covering rate and the date at which the sequence became public. If there is no extensive match with sequences other than ESTs or genomic DNA, or if the clone sequence brings substantial new information, such as encoding a protein resulting from alternative slicing of an mRNA coding for an already known protein, the sequence is kept. Examples of such cloned full length extended cDNAs containing sequences of interest are described in Example 28. Sequences resulting from chimera or double inserts as assessed by homology to other sequences are discarded during this procedure.
  • Example 27 The procedure described in Example 27 above was used to obtain the extended cDNAs described herein. Using this approach, the full length cDNA of SEQ ID NO:17 was obtained. This cDNA falls into the "EST-ext” category described above and encodes the signal peptide MKKVLLLITAILAVAVG (SEQ ID NO: 18) having a von Heijne score of 8.2.
  • the full length cDNA of SEQ ID NO:19 was also obtained using this procedure. This cDNA falls into the "EST-ext” category described above and encodes the signal peptide MWWFQQGLSFLPSALVIWTSA (SEQ ID NO:20) having a von Heijne score of 5.5.
  • the full length cDNA of SEQ ID NO:25 was also obtained using this procedure. This cDNA falls into the "new" category described above and encodes a signal peptide LVLTLCTLPLAVA (SEQ ID NO:26) having a von Heijne score of 10.1.
  • the full length cDNA of SEQ ID NO:27 was also obtained using this procedure. This cDNA falls into the "new" category described above and encodes a signal peptide LWLLFFLVTAIHA (SEQ ID NO:28) having a von Heijne score of 10.7.
  • nucleotides encoding both the signal peptide and the mature protein listed under the heading FCS location in Table IV
  • the location of the nucleotides in SEQ ID NO: 45 which encode the signal peptides listed under the heading SigPep Location in Table IV
  • the location of the nucleotides in SEQ ID NO: 45 which encode the mature proteins generated by cleavage of the signal peptide listed under the heading Mature Polypeptide Location in Table IV
  • stop codons listed under the heading Stop Codon Location in Table IV
  • polyA signals listed under the heading Poly A Signal Location in Table IV
  • polyA sites listed under the heading Poly A Site Location in Table IV
  • polypeptides encoded by the extended cDNAs were screened for the presence of known structural or functional motifs or for the presence of signatures, small amino acid sequences which are well conserved amongst the members of a protein family.
  • the conserved regions have been used to derive consensus patterns or matrices included in the PROSITE data bank, in particular in the file prosite-dat (Release 13.0 of November 1995. located at http://expasy.hcuge.ch/sprot/prosite.html.
  • Prosite_convert and prosite scan programs http://ulrec3.unil.ch/ftpserveur/prosite scan) were used to find signatures on the extended cDNAs.
  • db shuffled The program used to shuffle protein sequences (db shuffled) and the program used to determine the statistics for each pattern in the protein data banks (prosite statistics) are available on the ftp site http://utrec3.unil.ch//ftpserveur/prosite scan.
  • Table V lists the sequence identification number of the polypeptides of SEQ ID NO 146, the location of the amino acid residues of SEQ ID NO 146 in the full length polypeptide (second column), the location of the amino acid residues of SEQ ID NO 146 in the signal peptides (third column), and the location of the amino acid residues of SEQ ID NO 146 in the mature polypeptide created by cleaving the signal peptide from the full length polypeptide (fourth column).
  • nucleotide sequences of the sequences of SEQ ID NO: 45 and the amino acid sequence encoded by SEQ 10 NOs: 45 are provided in the appended sequence listing. In some instances, the sequences are preliminary and may include some incorrect or ambiguous sequences or amino acids.
  • the sequences of SEQ ID NO: 45 can readily be screened for any errors therein and any sequence ambiguities can be resolved by resequencing a fragment containing such errors or ambiguities on both strands. Nucleic acid fragments for resolving sequencing errors or ambiguities may be obtained from the deposited clones or can be isolated using the techniques described herein.
  • ambiguities or errors may be facilitated by using primers which hybridize to sequences located close to the ambiguous or erroneous sequences.
  • the primers may hybridize to sequences within 50-75 bases of the ambiguity or error.
  • the corresponding corrections can be made in the protein sequences encoded by the DNA containing the error or ambiguity.
  • ambiguities in the sequence of SEQ ID NO: 131 were resolved.
  • the amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein, and determining its sequence.
  • amino acid sequences For each amino acid sequence. Applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing. Some of the amino acid sequences may contain "Xaa” designators. These "Xaa” designators indicate either (1) a residue which cannot be identified because of nucleotide sequence ambiguity or (2) a stop codon in the determined sequence where Applicants believe one should not exist (if the sequence were determined more accurately).
  • Cells containing the extended cDNAs (SEQ ID NOs: 40-140 and 242-377) described herein in the vector pED6dpc2. are maintained in permanent deposit by the inventors at Genset. S.A., 24 Rue Royale, 75008 Paris, France.
  • SEQ ID NO: 45 SEQ ID NO: 45.
  • Table VII provides the internal designation number assigned to SEQ ID NO 45 and 146 and indicates whether the sequence is a nucleic acid sequence or a protein sequence.
  • Each extended cDNA can be removed from the pED6dpc2 vector in which it was deposited by performing a Notl. PstI double digestion to produce the appropriate fragment for each clone.
  • the proteins encoded by the extended cDNAs may also be expressed from.the promoter in pED6dpc2.
  • Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
  • oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The design of the oligonucleotide probe should preferably follow these parameters:
  • the oligonucleotide should preferably be labeled with (-[ 32 P]ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantified by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4X10 6 dpm/pmole.
  • the bacterial culture containing the pool of full-length clones should preferably be thawed and 100 ⁇ l of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 ug/ml.
  • the culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth.
  • Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 ⁇ g/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
  • Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
  • the filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 pg/ml of yeast RNA, and 10 mM EDTA (approximately 10 ml per 150 mm lilter).
  • 6X SSC 20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 pg/ml of yeast RNA, and 10 mM EDTA (approximately 10 ml per 150 mm lilter).
  • the probe is then added to the hybridization mix at a concentration greater than or equal to 1X10 5 dpm/mL.
  • the filter is then preferably incubated at 65°C with gentle agitation overnight.
  • the filter is then preferably washed in 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional.
  • the filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed.
  • the positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures.
  • the clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
  • plasmid DNA obtained using these procedures may then be manipulated using standard cloning techniques familiar to those skilled in the art.
  • a PCR can be done with primers designed at both ends of the extended cDNA insertion.
  • a PCR reaction may be conducted using a primer having the sequence GGCCATACACTTGAGTGAC (SEQ ID NO:38) and a primer having the sequence ATATAGACAAACGCACACC (SEQ. ID. NO:39).
  • the PCR product which corresponds to the extended cDNA can then be manipulated using standard cloning techniques familiar to those skilled in the art.
  • PCR based methods for obtaining extended cDNAs traditional hybridization based methods may also be employed. These methods may also be used to obtain the genomic DNAs which encode the mRNAs from which the 5' ESTs were derived, mRNAs corresponding to the extended cDNAs, or nucleic acids which are homologous to extended cDNAs or 5' ESTs.
  • Example 29 below provides an example of such methods.
  • a full length cDNA library can be made using the strategies described in Examples 13, 14, 15, and 16 above by replacing the random nonamer used in Example 14 with an oligo-dT primer.
  • the oligonucleotide of SEQ ID NO:14 may be used.
  • a cDNA library or genomic DNA library may be obtained from a commercial source or made using techniques familiar to those skilled in the art.
  • the library includes cDNAs which are derived from the mRNA corresponding to a 5' EST or which have homology to an extended cDNA or 5' EST.
  • the cDNA library or genomic DNA library is hybridized to a detectable probe comprising at least 10 consecutive nucleotides from the 5' EST or extended cDNA using conventional techniques.
  • the probe comprises at least 12, 15, or 17 consecutive nucleotides from the 5' EST or extended cDNA. More preferably, the probe comprises at least 20-30 consecutive nucleotides from the 5' EST or extended cDNA.
  • the probe comprises at least 30 nucleotides from the 5' EST or extended cDNA. In other embodiments, the probe comprises at least 40, at least 50, at least 75, at least 100, at least 150, or at least 200 consecutive nucleotides from the 5' EST or extended cDNA.
  • a probe comprising at least 10 consecutive nucleotides from the 5' EST or extended cDNA is labeled with a detectable label such as a radioisotope or a fluorescent molecule.
  • the probe comprises at least 12, 15, or 17 consecutive nucleotides from the 5' EST or extended cDNA. More preferably, the probe comprises 20-30 consecutive nucleotides from the 5' EST or extended cDNA. In some embodiments, the probe comprises more than 30 nucleotides from the 5' EST or extended cDNA.
  • the probe comprises at least 40, at least 50, at least 75, at least 100, at least 150, or at least 200 consecutive nucleotides from the 5' EST or extended cDNA.
  • Techniques for labeling the probe include phosphorylation with polynucleotide kinase, nick translation, in vitro transcription, and non-radioactive techniques.
  • the cDNAs or genomic DNAs in the library are transferred to a nitrocellulose or nylon filter and denatured. After incubation of the filter with a blocking solution, the filter is contacted with the labeled probe and incubated for a sufficient amount of time for the probe to hybridize to cDNAs or genomic DNAs containing a sequence capable of hybridizing to the probe.
  • the melting temperature of the probe may be calculated using the following formulas:
  • Prehybridization may be carried out in 6X SSC, 5X Denhardt's reagent, 0.5% SDS, 100 ⁇ g denatured fragmented salmon sperm DNA or 6X SSC, 5X Denhardt's reagent, 0.5% SDS, 100 ⁇ g denatured fragmented salmon sperm DNA, 50% formamide.
  • 6X SSC 6X SSC
  • 5X Denhardt's reagent 0.5% SDS
  • 100 ⁇ g denatured fragmented salmon sperm DNA 50% formamide.
  • the formulas for SSC and Denhardt's solutions are listed in Sambrook et al., supra.
  • Hybridization is conducted by adding the detectable probe to the prehybridization solutions listed above. Where the probe comprises double stranded DNA, it is denatured before addition to the hybridization solution. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to extended cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto. For probes over 200 nucleotides in length, the hybridization may be carried out at 15-25°C below the Tm. For shorter probes, such as oligonucleotide probes, the hybridization may be conducted at 15-25°C below the Tm. Preferably, for hybridizations in 6X SSC, the hybridization is conducted at approximately 68°C. Preferably, for hybridizations in 50% formamide containing solutions, the hybridization is conducted at approximately 42°C.
  • Extended cDNAs, nucleic acids homologous to extended cDNAs or 5' ESTs, or genomic DNAs which have hybridized to the probe are identified by autoradiography or other conventional techniques.
  • the above procedure may be modified to identify extended cDNAs, nucleic acids homologous to extended cDNAs, or genomic DNAs having decreasing levels of homology to the probe sequence.
  • less stringent conditions may be used.
  • the hybridization temperature may be decreased in increments of 5°C from 68°C to 42°C in a hybridization buffer having a Na+ concentration of approximately 1 M.
  • the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be "moderate” conditions above 50°C and "low” conditions below 50°C.
  • the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C.
  • concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe.
  • the filter may be washed with 6X SSC, 0.5% SDS at 50°C. These conditions are considered to be “moderate” conditions above 25% formamide and "low” conditions below 25% formamide.
  • Extended cDNAs, nucleic acids homologous to extended cDNAs, or genomic DNAs which have hybridized to the probe are identified by autoradiography.
  • the level of homology between the hybridized nucleic acid and the extended cDNA or 5' EST used as the probe may readily be determined.
  • the nucleotide sequences of the hybridized nucleic acid and the extended cDNA or 5'EST from which the probe was derived are compared.
  • nucleic acids having at least 95% nucleic acid homology to the extended cDNA or 5'EST from which the probe was derived may be obtained and identified.
  • nucleic acids having at least 90%, at least 85%, at least 80% or at least 75% homology to the extended cDNA or 5'EST from which the probe was derived may be further determined using BLAST2N; parameters may be adapted depending on the sequence length and degree of homology studied. In such comparisons, the default parameters or the parameters listed in Tables II and III may be used.
  • the amino acid sequence encoded by the extended cDNA or 5' EST is compared to the amino acid sequence encoded by the hybridizing nucleic acid. Homology is determined to exist when an amino acid sequence in the extended cDNA or 5' EST is closely related to an amino acid sequence in the hybridizing nucleic acid. A sequence is closely related when it is identical to that of the extended cDNA or 5' EST or when it contains one or more amino acid substitutions therein in which amino acids having similar characteristics have been substituted for one another.
  • nucleic acids encoding proteins having at least 95%, at least 90%, at least 85%, at least 80% or at least 75% homology to the proteins encoded by the extended cDNA or 5'EST from which the probe was derived Using the above methods and algorithms such as FASTA with parameters depending on the sequence length and degree of homology studied the level of homology may be determined. In determining the level of homology using FASTA, the default parameters or the parameters listed in Tables II or III may be used.
  • extended cDNAs may be prepared by obtaining mRNA from the tissue, cell, or organism of interest using mRNA preparation procedures utilizing poly A selection procedures or other techniques known to those skilled in the art.
  • a first primer capable of hybridizing to the poly A tail of the mRNA is hybridized to the mRNA and a reverse transcription reaction is performed to generate a first cDNA strand.
  • the first cDNA strand is hybridized to a second primer containing at least 10 consecutive nucleotides of the sequences of the 5' EST for which an extended cDNA is desired.
  • the primer comprises at least 12, 15, or 17 consecutive nucleotides from the sequences of the 5' EST. More preferably, the primer comprises 20-30 consecutive nucleotides from the sequences of the 5' EST. In some embodiments, the primer comprises more than 30 nucleotides from the sequences of the 5' EST.
  • the second primer used contains sequences located upstream of the translation initiation site. The second primer is extended to generate a second cDNA strand complementary to the first cDNA strand.
  • RTPCR may be performed as described above using primers from both ends of the cDNA to be obtained.
  • Extended cDNAs containing 5' fragments of the mRNA may be prepared by contacting an mRNA comprising the sequence of the 5' EST for which an extended cDNA is desired with a primer comprising at least 10 consecutive nucleotides of the sequences complementary to the 5' EST, hybridizing the primer to the mRNAs, and reverse transcribing the hybridized primer to make a first cDNA strand from the mRNAs.
  • the primer comprises at least 12, 15, or 17 consecutive nucleotides from the 5' EST. More preferably, the primer comprises 20-30 consecutive nucleotides from the 5' EST.
  • a second cDNA strand complementary to the first cDNA strand is synthesized.
  • the second cDNA strand may be made by hybridizing a primer complementary to sequences in the first cDNA strand to the first cDNA strand and extending the primer to generate the second cDNA strand.
  • the double stranded extended cDNAs made using the methods described above are isolated and cloned.
  • the extended cDNAs may be cloned into vectors such as plasmids or viral vectors capable of replicating in an appropriate host cell.
  • the host cell may be a bacterial, mammalian, avian, or insect cell.
  • kits for obtaining full length cDNAs such as the GeneTrapper (Cat. No. 10356-020, Gibco, BRL), may be used for obtaining full length cDNAs or extended cDNAs.
  • full length or extended cDNAs are prepared from mRNA and cloned into double stranded phagemids.
  • the cDNA library in the double stranded phagemids is then rendered single stranded by treatment with an endonuclease, such as the Gene II product of the phage F1, and Exonuclease III as described in the manual accompanying the GeneTrapper kit.
  • a biotinylated oligonucleotide comprising the sequence of a 5' EST, or a fragment containing at least 10 nucleotides thereof, is hybridized to the single stranded phagemids.
  • the fragment comprises at least 12, 15, or 17 consecutive nucleotides from the 5' EST. More preferably, the fragment comprises 20-30 consecutive nucleotides from the 5' EST. In some procedures, the fragment may comprise more than 30 consecutive nucleotides from the 5' EST.
  • the fragment may comprises at least 40, at least 50, at least 75, at least 100, at least 150, or at least 200 consecutive nucleotides from the 5' EST.
  • Hybrids between the biotinylated oligonucleotide and phagemids having inserts containing the 5' EST sequence are isolated by incubating the hybrids with streptavidin coated paramagnetic beads and retrieving the beads with a magnet. Thereafter, the resulting phagemids containing the 5' EST sequence are released from the beads and converted into double stranded DNA using a primer specific for the 5' EST sequence. The resulting double stranded DNA is transformed into bacteria. Extended cDNAs containing the 5' EST sequence are identified by colony PCR or colony hybridization.
  • a plurality of extended cDNAs containing full length protein coding sequences or sequences encoding only the mature protein remaining after the signal peptide is cleaved may be provided as cDNA libraries for subsequent evaluation of the encoded proteins or use in diagnostic assays as described below.
  • Extended cDNAs containing the full protein coding sequences of their corresponding mRNAs or portions thereof may be used to express the secreted proteins or portions thereof which they encode as described in Example 30 below.
  • the extended cDNAs may contain the sequences encoding the signal peptide to facilitate secretion of the expressed protein. It will be appreciated that a plurality of extended cDNAs containing the full protein coding sequences or portions thereof may be simultaneously cloned into expression vectors to create an expression library for analysis of the encoded proteins as described below.
  • nucleic acids containing the coding sequence for the proteins or portions thereof to be expressed are obtained as described in Examples 27-29 and cloned into a suitable expression vector.
  • the nucleic acids may contain the sequences encoding the signal peptide to facilitate secretion of the expressed protein.
  • the nucleic acid may comprise the sequence of SEQ ID NO 45 listed in Table IV and in the accompanying sequence listing.
  • the nucleic acid may comprise those nucleotides which make up the full coding sequence of the sequence of SEQ ID NO: 45 as defined in Table IV above.
  • the sequence of SEQ ID NO: 115 represents an alternatively spliced transcript of a previously identified mRNA.
  • any claims herein relating to nucleic acids containing the full coding sequence of SEQ ID NO. 45 is not to be construed as excluding any readily identifiable variations from or equivalents to the full coding sequences listed in Table IV
  • the scope of claims relating to polypeptides comprising the amino acid sequence of the full length polypeptide is not to be construed as excluding any readily identifiable variations from or equivalents to the sequences listed in Table V.
  • nucleic acid used to express the protein or portion thereof may comprise those nucleotides which encode the mature protein (i.e. the protein created by cleaving the signal peptide off) encoded by
  • SEQ ID NO: 45 as defined in Table IV above. It will be appreciated that should the extent of the sequence encoding the mature protein differ from that listed in Table IV as a result of a sequencing error, reverse transcription or amplification error, mRNA splicing, post-translational modification of the encoded protein, enzymatic cleavage of the encoded protein, or other biological factors, one skilled in the art would be readily able to identify the extent of the sequence encoding the mature protein in the sequence of SEQ ID NO. 45. Accordingly, the scope of any claims herein relating to nucleic acids containing the sequence encoding the mature protein encoded by SEQ ID No. 45 is not to be construed as excluding any readily identifiable variations from or equivalents to the sequences listed in Table IV.
  • claims relating to nucleic acids containing the sequence encoding the mature protein encompass equivalents to the sequences listed in Table IV. such as sequences encoding biologically active proteins resulting from post-translational modification, enzymatic cleavage, or other readily identifiable variations from or equivalents to the secreted protein in addition to cleavage of the signal peptide.
  • the extent of the mature polypeptide differ from that indicated in Table V as a result of any of the preceding factors, the scope of claims relating to polypeptides comprising the sequence of a mature protein included in the sequence of SEQ ID NOs. 146 is not to be construed as excluding any readily identifiable variations from or equivalents to the sequences listed in Table V.
  • claims relating to polypeptides comprising the sequence of the mature protein encompass equivalents to the sequences listed in Table IV, such as biologically active proteins resulting from post-translational modification, enzymatic cleavage, or other readily identifiable variations from or equivalents to the secreted proteins in addition to cleavage of the signal peptide. It will also be appreciated that should the biologically active form of the polypeptide included in the sequence of SEQ ID NOs.
  • nucleic acid encoding the biologically active form of the polypeptide differ from those identified as the mature polypeptide in Table V or the nucleotides encoding the mature polypeptide in Table IV as a result of a sequencing error, reverse transcription or amplification error, mRNA splicing, post-translational modification of the encoded protein, enzymatic cleavage of the encoded protein, or other biological factors, one skilled in the art would be readily able to identify the amino acids in the biologically active form of the polypeptides and the nucleic acids encoding the biologically active form of the polypeptides. In such instances, the claims relating to polypetides comprising the mature protein included in SEQ ID NO. 146 or nucleic acids comprising the nucleotides of SEQ ID NO. 45 encoding the mature protein shall not be construed to exclude any readily identifiable variations from the sequences listed in Table IV and Table V.
  • the nucleic acid used to express the protein or portion thereof may comprise those nucleotides which encode the signal peptide encoded by SEQ 10 NOs: 45 as defined in Table IV above.
  • the nucleic acid may encode a polypeptide comprising at least 10 consecutive amino acids of the sequence of SEQ ID NOs: 146.
  • the nucleic acid may encode a polypeptide comprising at least 15 consecutive amino acids of the sequence of SEQ ID NOs: 146.
  • the nucleic acid may encode a polypeptide comprising at least 25 consecutive amino acids of the sequence of SEQ ID NOs: 146.
  • the nucleic acid may encode a polypeptide comprising at least 60, at least 75, at least 100 or more than 100 consecutive amino acids of the sequence of SEQ ID Nos: 146.
  • the nucleic acids inserted into the expression vectors may also contain sequences upstream of the sequences encoding the signal peptide, such as sequences which regulate expression levels or sequences which confer tissue specific expression.
  • the nucleic acid encoding the protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology.
  • the expression vector may be any of the mammalian, yeast, insect or bacterial expression systems known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, MA), Stratagene (La Jolla, California), Promega (Madison, Wisconsin), and Invitrogen (San Diego, California). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence may be optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, et al., U.S. Patent No. 5,082,767 .
  • the following is provided as one exemplary method to express the proteins encoded ty the extended cDNAs corresponding to the 5' ESTs or the nucleic acids described above.
  • the methionine initiation codon for the gene and the poly A signal of the gene are identified. If the nucleic acid encoding the polypeptide to be expressed lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques.
  • this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using BglI and Sall restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene).
  • pXT1 contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection.
  • the vector includes the Herpes Simplex Thymidine Kinase promoter and the selectable neomycin gene.
  • the extended cDNA or portion thereof encoding the polypeptide to be expressed is obtained by PCR from the bacterial vector using oligonucleotide primers complementary to the extended cDNA or portion thereof and containing restriction endonuclease sequences for Pst I incorporated into the 5'primer and BgIII at the 5' end of the corresponding cDNA 3' primer, taking care to ensure that the extended cDNA is positioned in frame with the poly A signal.
  • the purified fragment obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXT1, now containing a poly A signal and digested with BgIII.
  • the ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, New York) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600ug/ml G418 (Sigma, St. Louis, Missouri). Preferably the expressed protein is released into the culture medium, thereby facilitating purification.
  • the extended cDNAs may be cloned into pED6dpc2 as described above.
  • the resulting pED6dpc2 constructs may be transfected into a suitable host cell, such as COS 1 cells. Methotrexate resistant cells are selected and expanded.
  • the protein expressed from the extended cDNA is released into the culture medium thereby facilitating purification.
  • Proteins in the culture medium are separated by gel electrophoresis. If desired, the proteins may be ammonium sulfate precipitated or separated based on size or charge prior to electrophoresis.
  • the expression vector lacking a cDNA insert is introduced into host cells or organisms and the proteins in the medium are harvested.
  • the secreted proteins present in the medium are detected using techniques such as Coomassie or silver staining or using antibodies against the protein encoded by the extended cDNA. Coomassie and silver staining techniques are familiar to those skilled in the art.
  • Antibodies capable of specifically recognizing the protein of interest may be generated using synthetic 15-mer peptides having a sequence encoded by the appropriate 5' EST, extended cDNA, or portion thereof.
  • the synthetic peptides are injected into mice to generate antibody to the polypeptide encoded by the 5' EST, extended cDNA, or portion thereof.
  • Secreted proteins from the host cells or organisms containing an expression vector which contains the extended cDNA derived from a 5' EST or a portion thereof are compared to those from the control cells or organism.
  • the presence of a band in the medium from the cells containing the expression vector which is absent in the medium from the control cells indicates that the extended cDNA encodes a secreted protein.
  • the band corresponding to the protein encoded by the extended cDNA will have a mobility near that expected based on the number of amino acids in the open reading frame of the extended cDNA.
  • the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.
  • the proteins expressed from host cells containing an expression vector containing an insert encoding a secreted protein or portion thereof can be compared to the proteins expressed in host cells containing the expression vector without an insert.
  • the presence of a band in samples from cells containing the expression vector with an insert which is absent in samples from cells containing the expression vector without an insert indicates that the desired protein or portion thereof is being expressed.
  • the band will have the mobility expected for the secreted protein or portion thereof.
  • the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.
  • the protein encoded by the extended cDNA may be purified using standard immunochromatography techniques.
  • a solution containing the secreted protein such as the culture medium or a cell extract, is applied to a column having antibodies against the secreted protein attached to the chromatography matrix.
  • the secreted protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins.
  • the specifically bound secreted protein is then released from the column and recovered using standard techniques.
  • the extended cDNA sequence or portion thereof may be incorporated into expression vectors designed for use in purification schemes employing chimeric polypeptides.
  • the coding sequence of the extended cDNA or portion thereof is inserted in frame with the gene encoding the other half of the chimera.
  • the other half of the chimera may be ⁇ -globin or a nickel binding polypeptide encoding sequence.
  • a chromatography matrix having antibody to ⁇ -globin or nickel attached thereto is then used to purify the chimeric protein.
  • Protease cleavage sites may be engineered between the ⁇ -globin gene or the nickel binding polypeptide and the extended cDNA or portion thereof.
  • the two polypeptides of the chimera may be separated from one another by protease digestion.
  • pSG5 which encodes rabbit ⁇ -globin.
  • Intron II of the rabbit ⁇ -globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression.
  • Standard methods are published in methods texts such as Davis et al., (Basic Methods in Molecular Biology, L.G. Davis, M.D. Dibner, and J.F. Battey, ed., Elsevier Press, NY, 1986 ) and many of the methods are available from Stratagene, Life Technologies, Inc., or Promega.
  • Polypeptide may additionally be produced from the construct using in vitro translation systems such as the In vitro Express TM Translation Kit (Stratagene).
  • the purified proteins may be tested for the ability to bind to the surface of various cell types as described in Example 31 below. It will be appreciated that a plurality of proteins expressed from these cDNAs may be included in a panel of proteins to be simultaneously evaluated for the activities specifically described below, as well as other biological roles for which assays for determining activity are available.
  • the proteins encoded by the 5' ESTs, extended cDNAs, or fragments thereof are cloned into expression vectors such as those described in Example 30.
  • the proteins are purified by size, charge, immunochromatography or other techniques familiar to those skilled in the art.
  • the proteins are labeled using techniques known to those skilled in the art.
  • the labeled proteins are incubated with cells or cell lines derived from a variety of organs or tissues to allow the proteins to bind to any receptor present on the cell surface. Following the incubation, the cells are washed to remove non-specifically bound protein.
  • the labeled proteins are detected by autoradiography. Alternatively, unlabeled proteins may be incubated with the cells and detected with antibodies having a detectable label, such as a fluorescent molecule, attached thereto.
  • Specificity of cell surface binding may be analyzed by conducting a competition analysis in which various amounts of unlabeled protein are incubated along with the labeled protein.
  • the amount of labeled protein bound to the cell surface decreases as the amount of competitive unlabeled protein increases.
  • various amounts of an unlabeled protein unrelated to the labeled protein is included in some binding reactions.
  • the amount of labeled protein bound to the cell surface does not decrease in binding reactions containing increasing amounts of unrelated unlabeled protein, indicating that the protein encoded by the cDNA binds specifically to the cell surface.
  • secreted proteins have been shown to have a number of important physiological effects and, consequently, represent a valuable therapeutic resource.
  • the secreted proteins encoded by the extended cDNAs or portions thereof made according to Examples 27-29 may be evaluated to determine their physiological activities as described below.
  • secreted proteins may act as cytokines or may affect cellular proliferation or differentiation.
  • Many protein factors discovered to date including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.
  • the activity of a protein described herein is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, 8aF3, MC9/G, M+ (preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7c and CMK.
  • the proteins encoded by the above extended cDNAs or portions thereof may be evaluated for their ability to regulate T cell or thymocyte proliferation in assays such as those described above or in the following references: Current Protocols in Immunology, Ed. by J.E. Coligan et al., Greene Publishing Associates and Wiley-Interscience ; Takai et at. J. Immunol, 137:3494-3500, 1986 . Bertagnolli et al. J. Immunol. 145:1706-1712, 1990 . Bertagnolli et al., Cellular Immunology 133:327-341, 1991 . Bertagnolli, et al. J. Immunol. 149:3778-3783, 1992 ; Bowman et al., J. Immunol. 152:1756-1761, 1994 .
  • the proteins encoded by the cDNAs may also be assayed for the ability to regulate the proliferation and differentiation of hematopoietic or lymphopoietic cells.
  • Many assays for such activity are familiar to those skilled in the art, including the assays in the following references: Bottomly, K., Davis, LS. and Lipsky, P.E., Measurement of Human and Murine Interleukin 2 and Interleukin 4, Current Protocols in Immunology., J.E. Coligan et al. Eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991 ; deVries et al., J. Exp. Med.
  • the proteins encoded by the cDNAs may also be assayed for their ability to regulate T-cell responses to antigens.
  • Many assays for such activity are familiar to those skilled in the art, including the assays described in the following references: Chapter 3 (In Vitro Assays for Mouse lymphocyte Function), Chapter 6 (Cytokines and Their Cellular Receptors) and Chapter 7, (Immunologic Studies in Humans) in Current Protocols in Immunology, J.E. Coligan et al. Eds. Greene Publishing Associates and Wiley-Interscience ; Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980 ; Weinberger et al., Eur. J. Immun. 11:405-411, 1981 ; Takai et al., J. Immunol. 137:3494-3500, 1986 ; Takai et al., J. Immunol. 140:508-512, 1988 .
  • proteins which exhibit cytokine, cell proliferation, or cell differentiation activity may then be formulated as pharmaceuticals and used to treat clinical conditions in which induction of cell proliferation or differentiation is beneficial.
  • genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.
  • the proteins encoded by the cDNAs may also be evaluated for their effects as immune regulators.
  • the proteins may be evaluated for their activity to influence thymocyte or splenocyte cytotoxicity.
  • Numerous assays for such activity are familiar to those skilled in the art including the assays described in the following references: Chapter 3 (In Vitro Assays for Mouse Lymphocyte Function 3.1-3.19) and Chapter 7 (Immunologic studies in Humans) in Current Protocols in Immunology, J.E. Coligan et al. Eds, Greene Publishing Associates and Wiley-Interscience ; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981 ; Herrmann et al., J. Immunol.
  • the proteins encoded by the cDNAs may also be evaluated for their effects on T-cell dependent immunoglobulin responses and isotype switching. Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Maliszewski, J. Immunol. 144:3028-3033, 1990 ; Mond, J.J. and Brunswick, M Assays for B Cell Function: In vitro Antibody Production, Vol 1 pp. 3.8.1-3.8.16 in Current Protocols in Immunology. J.E. Coligan et al Eds., John Wiley and Sons, Toronto. 1994 .
  • the proteins encoded by the cDNAs may also be evaluated for their effect on immune effector cells, including their effect on Th1 cells and cytotoxic lymphocytes.
  • Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Chapter 3 (In Vitro Assays for Mouse Lymphocyte function 3.1-3.19) and Chapter 7 (Immunologic Studies in Humans) in Current Protocols in Immunology, J.E. Coligan et al. Eds., Greene Publishing Associates and Wiley-Interscience ; Takai et al., J. Immunol. 137:3494-3500, 1986 ; Takai et al.; J. Immunol. 140:508-512, 1988 : Bertagnolli et al., J. Immunol. 149:3778-3783, 1992 .
  • the proteins encoded by the cDNAs may also be evaluated for their effect on dendritic cell mediated activation of naive T-cells.
  • Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Guery et al., J.
  • the proteins encoded by the cDNAs may also be evaluated for their influence on the lifetime of lymphocytes.
  • Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Darzynkiewicz et al., Cytometry 13:795-808, 1992 ; Gorczyca et al., Leukemia 7:659-670, 1993 ; Gorczyca et al., Cancer Research 53:1945-1951, 1993 ; Itoh et al., Cell 66:233-243, 1991 ; Zacharchuk, Journal of Immunology 145:4037-4045, 1990 ; Zamai et al., Cytometry 14:891-897, 1993 ; Gorczyca et al., International Journal of Oncology 1:639-648, 1992 .
  • Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994 ; Fine et al.. Cellular immunology 155: 111-122, 1994 ; Galy et al., Blood 85:2770-2778, 1995 ; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991 .
  • Those proteins which exhibit activity as immune system regulators activity may then be formulated as pharmaceuticals and used to treat clinical conditions in which regulation of immune activity is beneficial.
  • the protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SCID severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.
  • infectious diseases caused by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis.
  • a protein may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
  • a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems.
  • Other conditions, in which immune suppression is desired may also be treatable using a protein.
  • T-cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating of preventing one or more antigen functions (including without limitation 8 lymphocyte antigen functions (such as, for example, 87)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a 87 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having 87-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another 8 lymphocyte antigen (e.g., 87-1, B7-3) or blocking antibody
  • a molecule which inhibits or blocks interaction of a 87 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having 87-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another 8 lymphocyte antigen (e.g., 87-1, B7-3) or blocking antibody)
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
  • the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
  • Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and renogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992 ) and Turka et al., Proc. Natl. Acad. Sci USA. 89:11102-11105 (1992 ).
  • murine models of GVHD see Paul ed.. Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847 ) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
  • Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.
  • Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • Administration of reagents which block costimulation of T cells by disrupting receptor ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.
  • blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/pr/pr mice or NZB hybrid mice, murine autoimmuno collagen arthritis, diabetes mellitus in OD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856 ).
  • Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory form of B lymphocyte antigens systemically.
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to T cells in vivo, thereby activating the T cells.
  • up regulation or enhancement of antigen function may be useful in the induction of tumor immunity.
  • Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides.
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-1-like activity and/or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acids encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I a chain protein and ⁇ 2 macroglobulin protein or an MHC class II a chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • nucleic acids encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I a chain protein and ⁇ 2 macroglobulin protein or an MHC class II a chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.
  • the proteins encoded by the extended cDNAs or portions thereof may also be evaluated for their hematopoiesis regulating activity. For example, the effect of the proteins on embryonic stem cell differentiation may be evaluated. Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Johansson et al. Cellular Biology 15:141-151, 1995 ; Keller et al., Molecular and Cellular Biology 13:473-486, 1993 ; McClanahan et al., Blood 81:2903-2915, 1993 .
  • the proteins encoded by the extended cDNAs or portions thereof may also be evaluated for their influence on the lifetime of stem cells and stem cell differentiation.
  • Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Freshney, M.G. Methylcellulose Colony Forming Assays, in Culture of Hematopoietic Cells. R.I. Freshney, et al. Eds. pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994 ; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992 ; McNiece, I.K. and Briddell, R.A.
  • hematopoiesis regulatory activity may then be formulated as pharmaceuticals and used to treat clinical conditions in which regulation of hematopoeisis is beneficial.
  • a protein may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
  • Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e..
  • the proteins encoded by the extended cDNAs or portions thereof may also be evaluated for their effect on tissue growth.
  • Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in International Patent Publication No. WO95/16035 , International Patent Publication No. WO95/05846 and International Patent Publication No. WO91/07491 .
  • Assays for wound healing activity include, without limitation, those described in: Winter. Epidermal Wound Healing, pps. 71-112 (Maibach, H1 and Rovee, DT, eds.). Year Book Medical Publishers, Inc., Chicago , as modified by Eaglstein and Mertz. J. Invest. Dermatol 71:382-84 (1978 ).
  • Those proteins which are involved in the regulation of tissue growth may then be formulated as pharmaceuticals and used to treat clinical conditions in which regulation of tissue growth is beneficial.
  • a protein also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound heating and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
  • a protein which induces cartilage and/or bone growth in circumstances where bone is not normally formed has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a protein may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints.
  • De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • a protein may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
  • a protein may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • tissue regeneration activity that may be attributable to a protein is tendon/ligament formation.
  • Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
  • compositions may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.
  • the compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.
  • a protein may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e.. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease. Huntington's disease, amyotrophic lateral sclerosis, and Shy-Dragef syndrome. Further conditions which may be treated include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein.
  • Proteins may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
  • a protein may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium) muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to generate.
  • a protein may also exhibit angiogenic activity.
  • a protein may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokinc damage.
  • a protein may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
  • genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.
  • the proteins encoded by the extended cDNAs or portions thereof may also be evaluated for their ability to regulate reproductive hormones, such as follicle stimulating hormone.
  • reproductive hormones such as follicle stimulating hormone.
  • Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Vale et al., Endocrinology 91:562-572, 1972 ; Ling et al., Nature 321:779-782, 1986 ; Vale et al., Nature 321:776-779, 1986 ; Mason et al., Nature 318:659-663, 1985 ; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986 .
  • Those proteins which exhibit activity as reproductive hormones or regulators of cell movement may then be formulated as pharmaceuticals and used to treat clinical conditions in which regulation of reproductive hormones or cell movement are beneficial.
  • a protein may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins are characterized by their ability to stimulate the release of folic stimulating hormone (FSH).
  • FSH follicle stimulating hormone
  • FSH folic stimulating hormone
  • a protein alone or in heterodimers with a member of the inhibin a family may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.
  • the protein as a homodimer or as a heterodimer with other protein subunits of the inhibin-B group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885 .
  • a protein may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.
  • the proteins encoded by the extended cDNAs or portions thereof may also be evaluated for chemotactic/chemokinetic activity.
  • a protein may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, cosinophils, epithelial and/or endothelial cells.
  • Chemotactic and chmekinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
  • a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
  • the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
  • the activity of a protein of the invention may, among other means, be measured by the following methods: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhension of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub.
  • the proteins encoded by the extended cDNAs or portions thereof may also be evaluated for their effects on blood clotting.
  • Numerous assays for such activity are familiar to those skilled in the art, including the assays disclosed in the following references: Linet et al. J. Clin. Pharmacol. 26:131-140, 1986 ; Burdick et al., Thrombosis Res. 45:413-419, 1987 ; Humphrey et al., Fibrinolysis 5:71-79 (1991 ); Schaub, Prostaglandins 35:467-474, 1988 .
  • a protein may also exhibit hemostatic or thrombolytic activity.
  • a protein is expected to be useful in treatment of various coagulations disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
  • a protein may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g. stroke).
  • genes encoding these proteins or nucleic acids regulating the expression of these proteins may be introduced into appropriate host cells to increase or decrease the expression of the proteins as desired.
  • the proteins encoded by the extended cDNAs or a portion thereof may also be evaluated for their involvement in receptor/ligand interactions. Numerous assays for such involvement are familiar to those skilled in the art, including the assays disclosed in the following references: Chapter 7.28 (Measurement of Cellular Adhesion under Static Conditions 7.28.1-7.28.22) in Current Protocols in Immunology, J.E. Coligan et al. Eds. Greene Publishing Associates and Wiley-Interscience ; Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987 ; Bierer et al., J. Exp. Med. 168:1145-1156, 1988 ; Rosenstein et al., J.
  • the proteins may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
  • receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune respones).
  • Receptors and ligands are also useful for screening of potential peptide or small molecules inhibitors of the relevant receptor/ligand interaction.
  • a protein including, without limitation, fragments of receptors and ligands
  • the proteins encoded by the extended cDNAs or a portion thereof may also be evaluated for anti-inflammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
  • Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)),ischemia reperfusioninury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • the proteins encoded by the extended cDNAs or a portion thereof may also be evaluated for tumor inhibition activity.
  • a protein may exhibit other anti-tumor activities.
  • a protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC).
  • a protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, climinating or inhibiting factors, agents or cell types which promote tumor growth.
  • a protein may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites: effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or climination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of
  • Proteins which interact with the polypeptides encoded by extended cDNAs or portions thereof, such as receptor proteins, may be identified using two hybrid systems such as the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1, Clontech). As described in the manual accompanying the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1, Clontech), the extended cDNAs or portions thereof, are inserted into an expression vector such that they are in frame with DNA encoding the DNA binding domain of the yeast transcriptional activator GAL4.
  • cDNAs in a cDNA library which encode proteins which might interact with the polypeptides encoded by the extended cDNAs or portions thereof are inserted into a second expression vector such that they are in frame with DNA encoding the activation domain of GAL4.
  • the two expression plasmids are transformed into yeast and the yeast are plated on selection medium which selects for expression of selectable markers on each of the expression vectors as well as GAL4 dependent expression of the HIS3 gene.
  • Transformants capable of growing on medium lacking histidine are screened for GAL4 dependent lacZ expression. Those cells which are positive in both the histidine selection and the lacZ assay contain plasmids encoding proteins which interact with the polypeptide encoded by the extended cDNAs or portions thereof.
  • the system described in Lustig et al., Methods in Enzymology 283: 83-99 (1997 ) may be used for identifying molecules which interact with the polypeptides encoded by extended cDNAs.
  • in vitro transcription reactions are performed on a pool of vectors containing extended cDNA inserts cloned downstream of a promoter which drives in vitro transcription.
  • the resulting pools of mRNAs are introduced into Xenopus laevis oocytes.
  • the oocytes are then assayed for a desired acitivity.
  • pooled in vitro transcription products produced as described above may be translated in vitro.
  • the pooled in vitro translation products can be assayed for a desired activity or for interaction with a known polypeptide.
  • Proteins or other molecules interacting with polypeptides encoded by extended cDNAs can be found by a variety of additional techniques.
  • affinity columns containing the polypeptide encoded by the extended cDNA or a portion thereof can be constructed.
  • the affinity column contains chimeric, proteins in which the protein encoded by the extended cDNA or a portion thereof is fused to glutathione S-transferase, A mixture of cellular proteins or pool of expressed proteins as described above and is applied to the affinity column. Proteins interacting with the polypeptide attached to the column can then be isolated and analyzed on 2-D electrophoresis gel as described in Ramunsen et al. Electrophoresis, 18, 588-598 (1997 ). Alternatively, the proteins retained on the affinity column can be purified by electrophoresis based methods and sequenced. The same method can be used to isolate antibodies, to screen phage display products, or to screen phage display human antibodies.
  • Proteins interacting with polypeptides encoded by extended cDNAs or portions thereof can also be screened by using an Optical Biosensor as described in Edwards & Leatherbarrow, Analytical Biochemistry, 246, 1-6 (1997 ).
  • the main advantage of the method is that it allows the determination of the association rate between the protein and other interacting molecules. Thus, it is possible to specifically select interacting molecules with a high or low association rate.
  • a target molecule is linked to the sensor surface (through a carboxymethl dextran matrix) and a sample of test molecules is placed in contact with the target molecules. The binding of a test molecule to the target molecule causes a change in the refractive index and/ or thickness.
  • the target molecule can be one of the polypeptides encoded by extended cDNAs or a portion thereof and the test sample can be a collection of proteins extracted from tissues or cells, a pool of expressed proteins, combinatorial peptide and/ or chemical libraries,or phage displayed peptides.
  • the tissues or cells from which the test proteins are extracted can originate from any species.
  • a target protein is immobilized and the test population is a collection of unique polypeptides encoded by the extended cDNAs or portions thereof.
  • microdialysis coupled to HPLC method described by Wang et al., Chromatographia, 44, 205-208(1997 ) or the affinity capillary electrophoresis method described by Busch et al., J. Chromatogr. 777:311-328 (1997 ), can be used.
  • U.S. Patent No. 5,654.150 may also be used to identify molecules which interact with the polypeptides encoded by the extended cDNAs.
  • pools of extended cDNAs are transcribed and translated in vitro and the reaction products are assayed for interaction with a known polypeptide or antibody.
  • the proteins expressed from the extended cDNAs or portions may be assayed for numerous activities in addition to those specifically enumerated above.
  • the expressed proteins may be evaluated for applications involving control and regulation of inflammation, tumor proliferation at metastasis, infection, or other clinical conditions.
  • the proteins expressed from the extended cDNAs or portions thereof may be useful as nutritional agents or cosmetic agents.
  • the proteins expressed from the extended cDNAs or portions thereof may be used to generate antibodies capable of specifically binding to the expressed protein or fragments thereof as described in Example 40 below.
  • the antibodies may be capable of binding a full length protein encoded by the sequence of SEQ ID NOs : 45, a mature protein encoded by the sequence of SEQ ID NO. 45, or a signal peptide encoded by the sequence of SEO 10 No. 45.
  • the antibodies may be capable of binding fragments of the proteins expressed from the extended cDNAs which comprise at least 10 amino acids of the sequence of SEO ID NO: 146.
  • the antibodies may be capable of binding fragments of the proteins expressed from the extended cDNAs which comprise at least 15 amino acids of the sequence of SEQ ID NO: 146.
  • the antibodies may be capable of binding fragments of the proteins expressed from the extended cDNAs which comprise at least 25 amino acids of the sequence of SEQ ID NOs: 146. In further embodiments, the antibodies may be capable of binding fragments of the proteins expressed from the extended cDNAs which comprise at least 40 amino acids of the sequence of SEO ID NO: 146.
  • Substantially pure protein or polypeptide is isolated from the transfected or transformed cells as described in Example 30.
  • concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml.
  • Monoclonal or polyclonal antibody to the protein can then be prepared as follows:
  • Monoclonal antibody to epitopes of any of the peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler, G. and Milstein. C., Nature 256:495 (1975 ) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media).
  • HAT media aminopterin
  • the successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.
  • Antibody producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as Elisa, as originally described by Engvall, E., Meth. Enzymol. 70:419 (1980 ), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2 .
  • Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom described above, which can be unmodified or modified to enhance immunogerocity.
  • Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species, For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant.
  • host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable.
  • An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971 ).
  • Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example. Ouchterlony, D. et al., Chap.19 in: Handbook of Experimental Immunology' O. Wier (ed) Blackwell (1973 ). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 ⁇ M). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology. 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980 ).
  • Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.
  • the antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.
  • the extended cDNAs described herein may be used as reagents in isolation procedures, diagnostic assays, and forensic procedures.
  • sequences from the extended cDNAs (or genomic DNAs obtainable therefrom) may be detectably labeled and used as probes to isolate other sequences capable of hybridizing to them.
  • sequences from the extended cDNAs (or genomic DNAs obtainable therefrom) may be used to design PCR primers to be used in isolation, diagnostic, or forensic procedures.
  • the extended cDNAs may be used to prepare PCR primers for a variety of applications, including isolation procedures for cloning nucleic acids capable of hybridizing to such sequences, diagnostic techniques and forensic techniques.
  • the PCR primers are at least 10 bases, and preferably at least 12, 15, or 17 bases in length. More preferably, the PCR primers are at least 20-30 bases in length. In some embodiments, the PCR primers may be more than 30 bases in length. It is preferred that the primer pairs have approximately the same G/C ratio, so that melting temperatures are approximately the same.
  • a variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see Molecular Cloning to Genetic Engineering White, B.A. Ed.
  • PCR primers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase. Pfu polymerase, or Vent polymerase.
  • a thermostable polymerase such as Taq polymerase. Pfu polymerase, or Vent polymerase.
  • the nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample.
  • the hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites.
  • Probes derived from extended cDNAs or portions thereof may be labeled with detectable labels familiar to those skilled in the art, including radioisotopes and non radioactive labels, to provide a detectable probe.
  • the detectable probe may be single stranded or double stranded and may be made using techniques known in the art, including in vitro transcription, nick translation, or kinase reactions.
  • a nucleic acid sample containing a sequence capable of hybridizing to the labeled probe is contacted with the labeled probe. If the nucleic acid in the sample is double stranded, it may be denatured prior to contacting the probe.
  • the nucleic acid sample may be immobilized on a surface such as a nitrocellulose or nylon membrane.
  • the nucleic acid sample may comprise nucleic acids obtained from a variety of sources, including genomic DNA, cDNA libraries, RNA, or tissue samples.
  • Procedures used to detect the presence of nucleic acids capable of hybridizing to the detectable probe include well known techniques such as Southern blotting, Northern blotting, dot blotting, colony hybridization, and plaque hybridization.
  • the nucleic acid capable of hybridizing to the labeled probe may be cloned into vectors such as expression vectors, sequencing vectors, or in vitro transcription vectors to facilitate the characterization and expression of the hybridizing nucleic acids in the sample.
  • vectors such as expression vectors, sequencing vectors, or in vitro transcription vectors to facilitate the characterization and expression of the hybridizing nucleic acids in the sample.
  • such techniques may be used to isolate and clone sequences in a genomic library or cDNA library which are capable of hybridizing to the detectable probe as described in Example 30 above.
  • PCR primers made as described in Example 41 above may be used in forensic analyses, such as the DNA fingerprinting techniques described in Examples 43.47 below. Such analyses may utilize detectable probes or primers based on the sequences of the extended cDNAs isolated using the 5' ESTs (or genomic DNAs obtainable therefrom).
  • DNA samples are isolated from forensic specimens of, for example, hair, semen, blood or skin cells by conventional methods.
  • Corresponding sequences are obtained from a test subject.
  • Each of these identification DNAs is then sequenced using standard techniques, and a simple database comparison determines the differences, if any, between the sequences from the subject and those from the sample.
  • Statistically significant differences between the suspect's DNA sequences and those from the sample conclusively prove a lack of identity. This lack of identity can be proven, for example, with only one sequence. Identity, on the other hand, should be demonstrated with a large number of sequences, all matching.
  • a minimum of 50 statistically identical sequences of 100 bases in length are used to prove identity between the suspect and the sample.
  • primers are prepared from a large number of sequences identified herein. Preferably. 20 to 50 different primers are used. These primers are used to obtain a corresponding number of PCR-generaled DNA segments from the individual in question in accordance with Example 41. Each of these DNA segments is sequenced, using the methods set forth in Example 43. The database of sequences generated through this procedure uniquely identifies the individual from whom the sequences were obtained. The same panel of primers may then be used at any later time to absolutely correlate tissue or other biological specimen with that individual.
  • Example 44 The procedure of Example 44 is repeated to obtain a panel of at least 10 amplified sequences from an individual and a specimen.
  • the panel contains at least 50 amplified sequences. More preferably, the panel contains 100 amplified sequences. In some embodiments, the panel contains 200 amplified sequences.
  • This PCR-generated DNA is then digested with one or a combination of, preferably, four base specific restriction enzymes. Such enzymes are commercially available and known to those of skill in the art. After digestion, the resultant gene fragments are size separated in multiple duplicate wells on an agarose gel and transferred to nitrocellulose using Southern blotting techniques well known to those with skill in the art. For a review of Southern blotting see Davis et at. (Basic Methods in Molecular Biology, 1986, Elsevier Press. pp 62-65 ).
  • a panel of probes based on the sequences of the extended cDNAs (or genomic DNAs obtainable therefrom), or fragments thereof of at least 10 bases, are radioactively or colorimetrically labeled using methods known in the art, such as nick translation or end labeling, and hybridized to the Southern blot using techniques known in the art (Davis et al., supra ).
  • the probe comprises at least 12, 15, or 17 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). More preferably, the probe comprises at least 20-30 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom).
  • the probe comprises more than 30 nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). In other embodiments, the probe comprises at least 40, at least 50, at least 75, at least 100, at least 150, or at least 200 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom).
  • At least 5 to 10 of these labeled probes are used, and more preferably at least about 20 or 30 are used to provide a unique pattern.
  • the resultant bands appearing from the hybridization of a large sample of extended cDNAs (or genomic DNAs obtainable therefrom) will be a unique identifier. Since the restriction enzyme cleavage will be different for every individual, the band pattern on the Southern blot will also be unique. Increasing the number of extended cDNA probes will provide a statistically higher level of confidence in the identification since there will be an increased number of sets of bands used for identification.
  • Another technique for identifying individuals using the extended cDNA sequences disclosed herein utilizes a dot blot hybridization technique.
  • Genomic DNA is isolated from nuclei of subject to be identified. Oligonucleotide probes of approximately 30 bp in length are synthesized that correspond to at least 10, preferably 50 sequences from the extended cDNAs or genomic DNAs obtainable therefrom. The probes are used to hybridize to the genomic DNA through conditions known to those in the art. The oligonucleotides are end labeled with P 32 using polynucleotide kinase (Pharmacia). Dot Blots are created by spotting the genomic DNA onto nitrocellulose or the like using a vacuum dot blot manifold (BioRad, Richmond California).
  • the nitrocellulose filter containing the genomic sequences is baked or UV linked to the filter, prehybridized and hybridized with labeled probe using techniques known in the art (Davis et al. supra ).
  • the 32 P labeled DNA fragments are sequentially hybridized with successively stringent conditions to detect minimal differences between the 30 bp sequence and the DNA.
  • Tetramethylammonium chloride is useful for identifying clones containing small numbers of nucleotide mismatches ( Wood et al., Proc. Natl. Acad. Sci. USA 82(6):1585-1588 (1985 )).
  • a unique pattern of dots distinguishes one individual from another individual.
  • Extended cDNAs or oligonucleotides containing at least 10 consecutive bases from these sequences can be used as probes in the following alternative fingerprinting technique.
  • the probe comprises at least 12, 15, or 17 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). More preferably, the probe comprises at least 20-30 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). In some embodiments, the probe comprises more than 30 nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). In other embodiments, the probe comprises at least 40, at least 50, at least 75, at least 100, at least 150, or at least 200 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom).
  • a plurality of probes having sequences from different genes are used in the alternative fingerprinting technique.
  • Example 47 below provides a representative alternative fingerprinting procedure in which the probes are derived from extended cDNAs.
  • 20-mer oligonucleotides are prepared from a large number, e.g. 50, 100, or 200, of extended cDNA sequences (or genomic DNAs obtainable therefrom) using commercially available oligonucleotide services such as Genset, Paris, France.
  • Cell samples from the test subject are processed for DNA using techniques well known to those with skill in the art.
  • the nucleic acid is digested with restriction enzymes such as EcoRI and Xbal. Following digestion, samples are applied to wells for electrophoresis.
  • the procedure as known in the art, may be modified to accommodate polyacrylamide electrophoresis, however in this example, samples containing 5 ug of DNA are loaded into wells and separated on 0.8% agarose gels. The gels are transferred onto nitrocellulose using standard Southern blotting techniques.
  • the antibodies generated in Examples 30 and 40 above may be used to identify the tissue type or cell species from which a sample is derived as described above.
  • tissue specific antigens are identified by the visualization of tissue specific antigens by means of antibody preparations according to Examples 30 and 40 which are conjugated, directly or indirectly to a detectable marker.
  • Selected labeled antibody species bind to their specific antigen binding partner in tissue sections, cell suspensions, or in extracts of soluble proteins from a tissue sample to provide a pattern for qualitative or semi-qualitative interpretation.
  • Antisera for these procedures must have a potency exceeding that of the native preparation, and for that reason, antibodies are concentrated to a mg/ml level by isolation of the gamma globulin fraction, for example, by ionexchange chromatography or by ammonium sulfate fractionation. Also, to provide the most specific antisera, unwanted antibodies, for example to common proteins, must be removed from the gamma globulin fraction, for example by means of insoluble immunoabsorbents, before the antibodies are labeled with the marker. Either monoclonal or heterologous antisera is suitable for either procedure.
  • Purified, high-titer antibodies, prepared as described above, are conjugated to a detectable marker, as described, for example, by Fudenberg, H., Chap. 26 in: Basic 503 Clinical Immunology. 3rd Ed. Lange, Los Altos, California (1980 ) or Rose, N. et al., Chap. 12 in: Methods in Immunodiagnosis, 2d Ed. John Wiley 503 Sons, New York (1980 ).
  • a fluorescent marker either fluorescein or rhodamine
  • antibodies can also be labeled with an enzyme that supports a color producing reaction with a substrate, such as horseradish peroxidase. Markers can be added to tissue-bound antibody in a second step, as described below.
  • the specific antitissue antibodies can be labeled with ferritin or other electron dense particles, and localization of the ferritin coupled antigen-antibody complexes achieved by means of an electron microscope.
  • the antibodies are radiolabeled, with, for example 125 I, and detected by overlaying the antibody treated preparation with photographic emulsion.
  • Preparations to carry out the procedures can comprise monoclonal or polyclonal antibodies to a single protein or peptide identified as specific to a tissue type, for example, brain tissue, or antibody preparations to several antigenically distinct tissue specific antigens can be used in panels, independently or in mixtures, as required.
  • Tissue sections and cell suspensions are prepared for immunohistochemical examination according to common histological techniques. Multiple cryostat sections (about 4 ⁇ m, unfixed) of the unknown tissue and known control, are mounted and each slide covered with different dilutions of the antibody preparation. Sections of known and unknown tissues should also be treated with preparations to provide a positive control, a negative control, for example, pre-immune sera, and a control for non-specific staining, for example, buffer.
  • Treated sections are incubated in a humid chamber for 30 min at room temperature, rinsed, then washed in buffer for 30-45 min. Excess fluid is blotted away, and the marker developed.
  • tissue specific antibody was not labeled in the first incubation, it can be labeled at this time in a second antibody-antibody reaction, for example, by adding fluorescein- or enzyme-conjugated antibody against the immunoglobulin class of the antiserum-producing species, for example, fluorescein labeled antibody to mouse IgG.
  • fluorescein- or enzyme-conjugated antibody against the immunoglobulin class of the antiserum-producing species for example, fluorescein labeled antibody to mouse IgG.
  • fluorescein labeled antibody to mouse IgG.
  • the antigen found in the tissues by the above procedure can be quantified by measuring the intensity of color or fluorescence on the tissue section, and calibrating that signal using appropriate standards.
  • tissue specific proteins and identification of unknown tissues from that procedure is carried out using the labeled antibody reagents and detection strategy as described for immunohistochemistry; however the sample is prepared according to an electrophoretic technique to distribute the proteins extracted from the tissue in an orderly array on the basis of molecular weight for detention.
  • a tissue sample is homogenized using a Virtis apparatus; cell suspensions are disrupted by Dounce homogenization or osmotic lysis, using detergents in either case as required to disrupt cell membranes, as is the practice in the art.
  • Insoluble cell components such as nuclei, microsomes, and membrane fragments are removed by ultracentrifugation, and the soluble protein-containing fraction concentrated if necessary and reserved for analysis.
  • a sample of the soluble protein solution is resolved into individual protein species by conventional SDS polyacrylamide electrophoresis as described, for example, by Davis, L. et al., Section 19-2 in: Basic Methods in Molecular Biology (P. Leder, ed), Elsevier, New York (1986 ), using a range of amounts of polyacrylamide in a set of gels to resolve the entire molecular weight range of proteins to be detected in the sample.
  • a size marker is run in parallel for purposes of estimating molecular weights of the constituent proteins.
  • Sample size for analysis is a convenient volume of from 5 to55 ⁇ l, and containing from about 1 to 100 ⁇ g protein.
  • a detectable label can be attached to the primary tissue antigen-primary antibody complex according to various strategies and permutations thereof.
  • the primary specific antibody can be labeled; alternatively, the unlabeled complex can be bound by a labeled secondary anti-IgG antibody.
  • either the primary or secondary antibody is conjugated to a biotin molecule, which can, in a subsequent step, bind an avidin conjugated marker.
  • enzyme labeled or radioactive protein A which has the property of binding to any IgG, is bound in a final step to either the primary or secondary antibody.
  • tissue specific antigen binding at levels above those seen in control tissues to one or more tissue specific antibodies, prepared from the gene sequences identified from extended cONA sequences, can identify tissues of unknown origin, for example, forensic samples, or differentiated tumor tissue that has metastasized to foreign bodily sites.
  • extended cDNAs may be mapped to their chromosomal locations.
  • Example 49 below describes radiation hybrid (RH) mapping of human chromosomal regions using extended cDNAs.
  • Example 50 describes a representative procedure for mapping an extended cONA (or a genomic DNA obtainable therefrom) to its location on a human chromosome.
  • Example 51 describes mapping of extended cDNAs (or genomic DNAs obtainable therefrom) on metaphase chromosomes by Fluorescence In Situ Hybridization (FISH).
  • FISH Fluorescence In Situ Hybridization
  • RH mapping is a somatic cell genetic approach that can be used for high resolution mapping of the human genome.
  • cell lines containing one or more human chromosomes are lethally irradiated, breaking each chromosome into fragments whose size depends on the radiation dose. These fragments are rescued by fusion with cultured rodent cells, yielding subclones containing different portions of the human genome.
  • This technique is described by Benham et al. (Genomics 4:509-517, 1989 ) and Cox et al., (Science 250:245-250, 1990 ). The random and independent nature of the subclones permits efficient mapping of any human genome marker.
  • Human DNA isolated from a panel of 80-100 cell lines provides a mapping reagent for ordering extended cONAs (or genomic DNAs obtainable therefrom).
  • extended cONAs or genomic DNAs obtainable therefrom.
  • the frequency of breakage between markers is used to measure distance, allowing construction of fine resolution maps as has been done using conventional ESTs ( Schuler et al., Science 274:540-546, 1996 ).
  • RH mapping has been used to generate a high-resolution whole genome radiation hybrid map of human chromosome 17q22-q25.3 across the genes for growth hormone (GH) and thymidine kinase (TK) ( Foster et al., Genomics 33:185-192, 1996 ), the region surrounding the Gorlin syndrome gene ( Obermayr et al., Eur. J. Hum. Genet.
  • Extended cONAs may be assigned to human chromosomes using PCR based methodologies.
  • oligonucleotide primer pairs are designed from the extended cONA sequence (or the sequence of a genomic DNA obtainable therefrom) to minimize the chance of amplifying through an intron.
  • the oligonucleotide primers are 18-23 bp in length and are designed for PCR amplification.
  • the creation of PCR primers from known sequences is well known to those with skill in the art. For a review of PCR technology see Erlich, H.A., PCR Technology: Principles and Applications for DNA Amplification. 1992. W.H. Freeman and Co., New York .
  • the primers are used in polymerase chain reactions (PCR) to amplify templates from total human genomic DNA.
  • PCR conditions are as follows: 60 ng of genomic DNA is used as a template for PCR with 80 ng of each oligonucleotide primer, 0.6 unit of Taq polymerase, and 1 ⁇ Cu of a 32 P-labeled deoxycytidine triphosphate.
  • the PCR is performed in a microplate thermocycler (Techne) under the following conditions: 30 cycles of 94°C, 1.4 min; 55°C, 2 min; and 72°C, 2 min; with a final extension at 72°C for 10 min.
  • the amplified products are analyzed on a 6% polyacrylamide sequencing gel and visualized by autoradiography.
  • the PCR reaction is repeated with DNA templates from two panels of human-rodent somatic cell hybrids, BIOS PCRable DNA (BIOS Corporation) and NIGMS Human-Rodent Somatic Cell Hybrid Mapping Panel Number 1 (NIGMS, Camden, NJ).
  • PCR is used to screen a series of somatic cell hybrid cell lines containing defined sets of human chromosomes for the presence of a given extended cDNA (or genomic DNA obtainable therefrom).
  • DNA is isolated from the somatic hybrids and used as starting templates for PCR reactions using the primer pairs from the extended cDNAs (or genomic DNAs obtainable therefrom). Only those somatic cell hybrids with chromosomes containing the human gene corresponding to the extended cONA (or genomic DNA obtainable therefrom) will yield an amplified fragment.
  • the extended cDNAs (or genomic DNAs obtainable therefrom) are assigned to a chromosome by analysis of the segregation pattern of PCR products from the somatic hybrid DNA templates.
  • the single human chromosome present in all cell hybrids that give rise to an amplified fragment is the chromosome containing that extended cONA (or genomic DNA obtainable therefrom).
  • the extended cDNAs (or genomic DNAs obtainable therefrom) may be mapped to individual chromosomes using FISH as described in Example 51 below.
  • Fluorescence in situ hybridization allows the extended cDNA (or genomic DNA obtainable therefrom) to be mapped to a particular location on a given chromosome.
  • the chromosomes to be used for fluorescence in situ hybridization techniques may be obtained from a variety of sources including cell cultures, tissues, or whole blood.
  • chromosomal localization of an extended cDNA is obtained by FISH as described by Cherif et al. (Proc. Natl. Acad Sci. U.S.A., 87:6639-6643, 1990 ).
  • Metaphase chromosomes are prepared from phytohemagglutinin (PHA)-stimulated blood cell donors. PHA-stimulated lymphocytes from healthy males are cultured for 72 h in RPMI-1640 medium. For synchronization, methotrexate (10 ⁇ M) is added for 17 h, followed by addition of 5-bromodeoxyuridine (5-BudR, 0.1 mM) for 6 h.
  • PHA phytohemagglutinin
  • Colcemid (1 ⁇ g/ml) is added for the last 15 min before harvesting the cells.
  • Cells are collected, washed in RPMI, incubated with a hypotonic solution of KCl (75 mM) at 37°C for 15 min and fixed in three changes of methanol:acetic acid (3:1).
  • the cell suspension is dropped onto a glass slide and air dried.
  • the extended cDNA (or genomic DNA obtainable therefrom) is labeled with biotin-16 dUTP by nick translation according to the manufacturer's instructions (Bethesda Research Laboratories, Bethesda, MD), purified using a Sephadex G-50 column (Pharmacia, Upssala, Sweden) and precipitated.
  • hybridization buffer 50% formamide, 2 X SSC. 10% dextran sulfate, 1 mg/ml sonicated salmon sperm DNA, pH 7) and the probe is denatured at 70°C for 5-10 min.
  • Slides kept at 20°C are treated for 1 h at 37°C with RNase A (100 ⁇ g/ml), rinsed three times in 2 X SSC and dehydrated in an ethanol series. Chromosome preparations are denatured in 70% formamide, 2 X SSC for 2 min at 70°C, then dehydrated at 4°C.
  • the slides are treated with proteinase K (10 ⁇ g/100 ml in 20 mM Tris-HCl, 2 mM CaCl 2 ) at 37°C for 8 min and dehydrated.
  • the hybridization mixture containing the probe is placed on the slide, covered with a coverslip, sealed with rubber cement and incubated overnight in a humid chamber at 37°C.
  • the biotinylated probe is detected by avidin-FITC and amplified with additional layers of biotinylated goat anti-avidin and avidin-FITC.
  • fluorescent R-bands are obtained as previously described (Cherif et al., supra. ). The slides are observed under a LEICA fluorescence microscope (DMRXA). Chromosomes are counterstained with propidium iodide and the fluorescent signal of the probe appears as two symmetrical yellow-green spots on both chromatids of the fluorescent R-band chromosome (red).
  • DMRXA LEICA fluorescence microscope
  • the extended cDNAs (or genomic DNAs obtainable therefrom) have been assigned to particular chromosomes using the techniques described in Examples 49-51 above, they may be utilized to construct a high resolution map of the chromosomes on which they are located or to identify the chromosomes in a sample.
  • Chromosome mapping involves assigning a given unique sequence to a particular chromosome as described above. Once the unique sequence has been mapped to a given chromosome, it is ordered relative to other unique sequences located on the same chromosome.
  • One approach to chromosome mapping utilizes a series of yeast artificial chromosomes (YACs) bearing several thousand long inserts derived from the chromosomes of the organism from which the extended cDNAs (or genomic DNAs obtainable therefrom) are obtained. This approach is described in Ramaiah Nagaraja et al. Genome Research 7:210-222. March 1997 . Briefly, in this approach each chromosome is broken into overlapping pieces which are inserted into the YAC vector.
  • the YAC inserts are screened using PCR or other methods to determine whether they include the extended cDNA (or genomic DNA obtainable therefrom) whose position is to be determined. Once an insert has been found which includes the extended cDNA (or genomic DNA obtainable therefrom), the insert can be analyzed by PCR or other methods to determine whether the insert also contains other sequences known to be on the chromosome or in the region from which the extended cDNA (or genomic DNA obtainable therefrom) was derived. This process can be repeated for each insert in the YAC library to determine the location of each of the extended cONAs (or genomic DNAs obtainable therefrom) relative to one another and to other known chromosomal markers. In this way, a high resolution map of the distribution of numerous unique markers along each of the organisms chromosomes may be obtained.
  • extended cDNAs may also be used to identify genes associated with a particular phenotype, such as hereditary disease or drug response.
  • This example illustrates an approach useful for the association of extended cONAs (or genomic DNAs obtainable therefrom) with particular phenotypic characteristics.
  • a particular extended cONA (or genomic DNA obtainable therefrom) is used as a test probe to associate that extended cONA (or genomic DNA obtainable therefrom) with a particular phenotypic characteristic.
  • Extended cDNAs (or genomic DNAs obtainable therefrom) are mapped to a particular location on a human chromosome using techniques such as those described in Examples 49 and 50 or other techniques known in the art.
  • a search of Mendelian Inheritance in Man (V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) reveals the region of the human chromosome which contains the extended cONA (or genomic DNA obtainable therefrom) to be a very gene rich region containing several known genes and several diseases or phenotypes for which genes have not been identified.
  • the gene corresponding to this extended cONA (or genomic DNA obtainable therefrom) thus becomes an immediate candidate for each of these genetic diseases.
  • PCR primers from the extended cONA are used to screen genomic DNA, mRNA or cONA obtained from the patients.
  • Extended cONAs (or genomic DNAs obtainable therefrom) that are not amplified in the patients can be positively associated with a particular disease by further analysis.
  • the PCR analysis may yield fragments of different lengths when the samples are derived from an individual having the phenotype associated with the disease than when the sample is derived from a healthy individual, indicating that the gene containing the extended cDNA may be responsible for the genetic disease.
  • the present extended cONAs may also be used to construct secretion vectors capable of directing the secretion of the proteins encoded by genes inserted in the vectors.
  • secretion vectors may facilitate the purification or enrichment of the proteins encoded by genes inserted therein by reducing the number of background proteins from which the desired protein must be purified or enriched.
  • Exemplary secretion vectors are described in Example 54 below.
  • the secretion vectors described herein include a promoter capable of directing gene expression in the host cell, tissue, or organism of interest.
  • promoters include the Rous Sarcoma Virus promoter, the SV40 promoter, the human cytomegalovirus promoter, and other promoters familiar to those skilled in the art.
  • a signal sequence from an extended cONA (or genomic DNA obtainable therefrom), such as the signal sequence in SEQ ID NO: 45 as defined in Table IV above, is operably linked to the promoter such that the mRNA transcribed from the promoter will direct the translation of the signal peptide.
  • the host cell tissue, or organism may be any cell, tissue, or organism which recognizes the signal peptide encoded by the signal sequence in the extended cDNA (or genomic DNA obtainable therefrom). Suitable hosts include mammalian cells, tissues or organisms, avian cells, tissues, or organisms, insect cells, tissues or organisms, or yeast.
  • the secretion vector contains cloning sites for inserting genes encoding the proteins which are to be secreted.
  • the cloning sites facilitate the cloning of the insert gene in frame with the signal sequence such that a fusion protein in which the signal peptide is fused to the protein encoded by the inserted gene is expressed from the mRNA transcribed from the promoter.
  • the signal peptide directs the extracellular secretion of the fusion protein.
  • the secretion vector may be DNA or RNA and may integrate into the chromosome of the host, be stably maintained as an extrachromosomal replicon in the host, be an artificial chromosome, or be transiently present in the host.
  • Many nucleic acid backbones suitable for use as secretion vectors are known to those skilled in the art, including retroviral vectors, SV40 vectors, Bovine Papilloma Virus vectors, yeast integrating plasmids, yeast episomal plasmids, yeast artificial chromosomes, human artificial chromosomes, P element vectors, baculovirus vectors, or bacterial plasmids capable of being transiently introduced into the host.
  • the secretion vector may also contain a polyA signal such that the polyA signal is located downstream of the gene inserted into the secretion vector.
  • the secretion vector is introduced into the host cell, tissue, or organism using calcium phosphate precipitation, DEAE. Dextran, electroporation, liposome-mediated transfection, viral particles or as naked DNA.
  • the protein encoded by the inserted gene is then purified or enriched from the supernatant using conventional techniques such as ammonium sulfate precipitation, immunoprecipitation, immunochromatography, size exclusion chromatography, ion exchange chromatography, and hplc.
  • the secreted protein may be in a sufficiently enriched or pure state in the supernatant or growth media of the host to permit it to be used for its intended purpose without further enrichment.
  • the signal sequences may also be inserted into vectors designed for gene therapy.
  • the signal sequence is operably linked to a promoter such that mRNA transcribed from the promoter encodes the signal peptide.
  • a cloning site is located downstream of the signal sequence such that a gene encoding a protein whose secretion is desired may readily be inserted into the vector and fused to the signal sequence.
  • the vector is introduced into an appropriate host cell. The protein expressed from the promoter is secreted extracellularly, thereby producing a therapeutic effect.
  • the extended cDNAs or 5' ESTs may also be used to clone sequences located upstream of the extended cDNAs or 5' ESTs which are capable of regulating gene expression, induing promoter sequences, enhancer sequences, and other upstream sequences which influence transcription or translation levels. Once identified and cloned, these upstream regulatory sequences may be used in expression vectors designed to direct the expression of an inserted gene in a desired spatial, temporal, developmental, or quantitative fashion.
  • Example 55 describes a method for cloning sequences upstream of the extended cDNAs or 5' ESTs.
  • Sequences derived from extended cDNAs or 5' ESTs may be used to isolate the promoters of the corresponding genes using chromosome walking techniques.
  • chromosome walking technique which utilizes the GenomeWalkerTM kit available from Clontech, five complete genomic DNA samples are each digested with a different restriction enzyme which has a 6 base recognition site and leaves a blunt end. Following digestion, oligonucleotide adapters are ligated to each end of the resulting genomic DNA fragments.
  • a first PCR reaction is performed according to the manufacturer's instructions using an outer adaptor primer provided in the kit and an outer gene specific primer.
  • the gene specific primer should be selected to be specific for the extended cONA or 5' EST of interest and should have a melting temperature, length, and location in the extended cDNA or' EST which is consistent with its use in PCR reactions.
  • Each first PCR reaction contains 5ng of genomic DNA, 5 ⁇ l of 10X Tth reaction buffer, 0.2 mM of each dNTP, 0.2 ⁇ M each of outer adaptor primer and outer gene specific primer, 1.1 mM of Mg(OAc) 2 , and 1 ⁇ l of the Tth polymerase 50X mix in a total volume of 50 ⁇ l.
  • the reaction cycle for the first PCR reaction is as follows: 1 min @ 94°C /2 sec @ 94°C, 3 min @ 72°C (7 cycles) / 2 sec @ 94°C, 3 min @ 67°C (32 cycles) / 5 min @ 67°C.
  • the product of the first PCR reaction is diluted and used as a template for a second PCR reaction according to the manufacturer's instructions using a pair of nested primers which are located internally on the amplicon resulting from the first PCR reaction.
  • a pair of nested primers which are located internally on the amplicon resulting from the first PCR reaction.
  • 5 ⁇ l of the reaction product of the first PCR reaction mixture may be diluted 180 times.
  • Reactions are made in a 50 ⁇ l volume having a composition identical to that of the first PCR reaction except the nested primers are used.
  • the first nested primer is specific for the adaptor, and is provided with the GenomeWalkerTM kit.
  • the second nested primer is specific for the particular extended cDNA or 5' EST for which the promoter is to be cloned and should have a melting temperature, length, and location in the extended cDNA or 5' EST which is consistent with its use in PCR reactions.
  • the reaction parameters of the second PCR reaction are as follows:1 min @ 94°C / 2 sec @ 94°C. 3 min @ 72°C (6 cycles) / 2 sec @ 94°C, 3 min @ 67°C (25 cycles) / 5 min @ 67°C
  • the product of the second PCR reaction is purified, cloned, and sequenced using standard techniques.
  • two or more human genomic DNA libraries can be constructed by using two or more restriction enzymes.
  • the digested genomic DNA is cloned into vectors which can be converted into single stranded, circular, or linear DNA.
  • a biotinylated oligonucleotide comprising at least 15 nucleotides from the extended cONA or 5' EST sequence is hybridized to the single stranded DNA. Hybrids between the biotinylated oligonucleotide and the single stranded DNA containing the extended cDNA or EST sequence are isolated as described in Example 29 above.
  • the single stranded DNA containing the extended cDNA or EST sequence is released from the beads and converted into double stranded DNA using a primer specific for the extended cONA or 5' EST sequence or a primer corresponding to a sequence included in the cloning vector.
  • the resulting double stranded DNA is transformed into bacteria.
  • DNAs containing the 5' EST or extended cDNA sequences are identified by colony PCR or colony hybridization.
  • prospective promoters and transcription start sites within the upstream sequences may be identified by comparing the sequences upstream of the extended cDNAs or 5' ESTs with databases containing known transcription start sites, transcription factor binding sites, or promoter sequences.
  • promoters in the upstream sequences may be identified using promoter reporter vectors as described in Example 56.
  • each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, ⁇ galactosidase, or green fluorescent protein.
  • the sequences upstream of the extended cDNAs or 5' ESTs are inserted into the cloning sites upstream of the reporter gene in both orientations and introduced into an appropriate host cell.
  • the level of reporter protein is assayed and compared to the level obtained from a vector which lacks an insert in the cloning site.
  • the presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert.
  • the upstream sequences can be cloned into vectors which contain an enhancer for augmenting transcription levels from weak promoter sequences. A significant level of expression above that observed with the vector lacking an insert indicates that a promoter sequence is present in the inserted upstream sequence.
  • Appropriate host cells for the promoter reporter vectors may be chosen based on the results of the above described determination of expression patterns of the extended cDNAs and ESTs. For example, if the expression pattern analysis indicates that the mRNA corresponding to a particular extended cDNA or 5' EST is expressed in fibroblasts, the promoter reporter vector may be introduced into a human fibroblast cell line.
  • Promoter sequences within the upstream genomic DNA may be further defined by constructing nested deletions in the upstream DNA using conventional techniques such as Exonuclease III digestion. The resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity. In this way, the boundaries of the promoters may be defined. If desired, potential individual regulatory sites within the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter individually or in combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into the cloning sites in the promoter reporter vectors.
  • Figure 8 provides a schematic description of the promoters isolated and the way they are assembled with the corresponding 5' tags.
  • the upstream sequences were screened for the presence of motifs resembling transcription factor binding sites or known transcription start sites using the computer program MatInspector release 2.0, August 1996.
  • Figure 9 describes the transcription factor binding sites present in each of these promoters.
  • the columns labeled matrice provides the name of the MatInspector matrix used.
  • the column labeled position provides the 5' postion of the promoter site. Numeration of the sequence starts from the transcription site as determined by matching the genomic sequence with the 5' EST sequence.
  • the column labeled "orientation” indicates the DNA strand on which the site is found, with the + strand being the coding strand as determined by matching the genomic sequence with the sequence of the 5' EST.
  • the column labeled “score” provides the MatInspector score found for this site.
  • the column labeled "length” provides the length of the site in nucleotides.
  • the column labeled "sequence” provides the sequence of the site found.
  • the promoters and other regulatory sequences located upstream of the extended cDNAs or 5' ESTs may be used to design expression vectors capable of directing the expression of an inserted gene in a desired spatial, temporal, developmental, or quantitative manner.
  • a promoter capable of directing the desired spatial; temporal, developmental, and quantitative patterns may be selected using the results of the expression analysis described in Example 26 above. For example, if a promoter which confers a high level of expression in muscle is desired, the promoter sequence upstream of an extended cDNA or 5' EST derived from an mRNA which is expressed at a high level in muscle, as determined by the method of Example 26, may be used in the expression vector.
  • the desired promoter is placed near multiple restriction sites to facilitate the cloning of the desired insert downstream of the promoter, such that the promoter is able to drive expression of the inserted gene.
  • the promoter may be inserted in conventional nucleic acid backbones designed for extrachromosomal replication, integration into the host chromosomes or transient expression.
  • Suitable backbones for the present expression vectors include retroviral backbones, backbones from eukaryotic episomes such as SV40 or Bovine Papilloma Virus, backbones from bacterial episomes, or artificial chromosomes.
  • the expression vectors also include a polyA signal downstream of the multiple restriction sites for directing the polyadenylation of mRNA transcribed from the gene inserted into the expression vector.
  • Sequences within the promoter region which are likely to bind transcription factors may be identified by homology to known transcription factor binding sites or through conventional mutagenesis or deletion analyses of reporter plasmids containing the promoter sequence. For example, deletions may be made in a reporter plasmid containing the promoter sequence of interest operably linked to an assayable reporter gene. The reporter plasmids carrying various deletions within the promoter region are transfected into an appropriate host cell and the effects of the deletions on expression levels is assessed. Transcription factor binding sites within the regions in which deletions reduce expression levels may be further localized using site directed mutagenesis, linker scanning analysis, or other techniques familiar to those skilled in the art.
  • Nucleic acids encoding proteins which interact with sequences in the promoter may be identified using one hybrid systems such as those described in the manual accompanying the Matchmaker One-Hybrid System kit avalilabe from Clontech (Catalog No. K1603-1). Briefly, the Matchmaker One-hybrid system is used as follows.
  • the target sequence for which it is desired to identify binding proteins is cloned upstream of a selectable reporter gene and integrated into the yeast genome. Preferably, multiple copies of the target sequences are inserted into the reporter plasmid in tandem.
  • the yeast are plated on selective media to select cells expressing the selectable marker linked to the promoter sequence.
  • the colonies which grow on the selective media contain genes encoding proteins which bind the target sequence.
  • the inserts in the genes encoding the fusion proteins are further characterized by sequencing.
  • the inserts may be inserted into expression vectors or in vitro transcription vectors. Binding of the polypeptides encoded by the inserts to the promoter DNA may be confirmed by techniques familiar to those skilled in the art, such as gel shift analysis or DNAse protection analysis.
  • the extended cDNAs may also be used in gene therapy strategies, including antisense and triple helix strategies as described in Examples 57 and 58 below.
  • antisense approaches nucleic acid sequences complementary to an mRNA are hybridized to the mRNA intracellularly, thereby blocking the expression of the protein encoded by the mRNA.
  • the antisense sequences may prevent gene expression through a variety of mechanisms. For example, the antisense sequences may inhibit the ability of ribosomes to translate the mRNA. Alternatively, the antisense sequences may block transport of the mRNA from the nucleus to the cytoplasm, thereby limiting the amount of mRNA available for translation.
  • antisense sequences may inhibit gene expression is by interfering with mRNA splicing.
  • the antisense nucleic acid may be incorporated in a ribozyme capable of specifically cleaving the target mRNA.
  • the antisense nucleic acid molecules to be used in gene therapy may be either DNA or RNA sequences. They may comprise a sequence complementary to the sequence of the extended cDNA (or genomic DNA obtainable therefrom).
  • the antisense nucleic acids should have a length and melting temperature sufficient to permit formation of an intracellular duplex having sufficient stability to inhibit the expression of the mRNA in the duplex.
  • Strategies for designing antisense nucleic acids suitable for use in gene therapy are disclosed in Green et al., Ann. Rev. Biochem. 55:569-597 (1986 ) and Izant and Weintraub, Cell 36:1007-1015 (1984 ).
  • antisense molecules are obtained from a nucleotide sequence encoding a protein by reversing the orientation of the coding region with respect to a promoter so as to transcribe the opposite strand from that which is normally transcribed in the cell.
  • the antisense molecules may be transcribed using in vitro transcription systems such as those which employ T7 or SP6 polymerase to generate the transcript.
  • Another approach involves transcription of the antisense nucleic acids in vivo by operably linking DNA containing the antisense sequence to a promoter in an expression vector.
  • oligonucleotides which are complementary to the strand normally transcribed in the cell may be synthesized in vitro.
  • the antisense nucleic acids are complementary to the corresponding mRNA and are capable of hybridizing to the mRNA to create a duplex.
  • the antisense sequences may contain modified sugar phosphate backbones to increase stability and make them less sensitive to RNase activity. Examples of modifications suitable for use in antisense strategies are described by Rossi et al., Pharmacol. Ther. 50(2):245-254. (1991 ).
  • antisense oligonucleotides complementary to the sequence of the extended cDNA (or genomic DNA obtainable therefrom) may be used.
  • stable and semi-stable antisense oligonucleotides described in International Application No. PCT WO94/23026 are used. In these moleucles, the 3' end or both the 3' and 5' ends are engaged in intramolecular hydrogen bonding between complementary base pairs. These molecules are better able to withstand exonuclease attacks and exhibit increased stability compared to conventional antisense oligonucleotides.
  • antisense oligodeoxynucleotides against herpes simplex virus types 1 and 2 described in International Application No. WO 95/04141 are also preferred examples.
  • the covalently cross-linked antisense oligonucleotides described in International Application No. WO 96/31523 are used.
  • These double- or single-stranded oligonucleotides comprise one or more, respectively, inter- or intra-oligonucleotide covalent cross-linkages, wherein the linkage consists of an amide bond between a primary amine group of one strand and a carboxyl group of the other strand or of the same strand, respectively, the primary amine group being directly substituted in the 2' position of the strand nucleotides monosaccharide ring, and the carboxyl group being carried by an aliphatic spacer group substituted on a nucleotide or nucleotide analog of the other strand or the same strand, respectively.
  • the antisense-oligodeoxynucleotides and oligonucleotides disclosed in International Application No. WO 92/18522 may also be used. These molecules are stable to degradation and contain at least one transcription control recognition sequence which binds to control proteins and are effective as decoys therefor. These molecules may contain "hairpin” structures, “dumbbell' structures, “modified dumbbell” structures, "cross-linked” decoy structures and “loop” structures.
  • oligonucleotide described in European Patent Application No. 0 572 287 A2 are used. These ligated oligonucleotide "dumbbells" contain the binding site for a transcription factor and inhibit expression of the gene under control of the transcription factor by sequestering the factor.
  • oligonucleotides Use of the closed antisense oligonucleotides disclosed in International Application No. WO 92/19732 is also contemplated. Because these molecules have no free ends, they are more resistant to degradation by exonucleases than are conventional oligonucleotides. These oligonucleotides may be multifunctional, interacting with several regions which are not adjacent to the target mRNA.
  • the appropriate level of antisense nucleic acids required to inhibit gene expression may be determined using in vitro expression analysis.
  • the antisense molecule may be introduced into the cells by diffusion, injection, infection or transfection using procedures known in the art.
  • the antisense nucleic acids can be introduced into the body as a bare or naked oligonucleotide, oligonucleotide encapsulated in lipid, oligonucleotide sequence encapsidated by viral protein, or as an oligonucleotide operably linked to a promoter contained in an expression vector.
  • the expression vector may be any of a variety of expression vectors known in the art, including retroviral or viral vectors, vectors capable of extrachromosomal replication, or integrating vectors.
  • the vectors may be DNA or RNA.
  • the antisense molecules are introduced onto cell samples at a number of different concentrations preferably between 1x10 -10 M to 1x10 -4 M. Once the minimum concentration that can adequately control gene expression is identified, the optimized dose is translated into a dosage suitable for use in vivo. For example, an inhibiting concentration in culture of 1x10 -7 translates into a dose of approximately 0.6 mg/kg bodyweight. Levels of oligonucleotide approaching 100 mg/kg bodyweight or higher may be possible after testing the toxicity of the oligonucleotide in laboratory animals. It is additionally contemplated that cells from the vertebrate are removed, treated with the antisense oligonucleotide, and reintroduced into the vertebrate.
  • the antisense oligonucleotide sequence is incorporated into a ribozyme sequence to enable the antisense to specifically bind and cleave its target mRNA.
  • ribozyme and antisense oligonucleotides see Rossi et al., supra .
  • the polypeptide encoded by the gene is first identified, so that the effectiveness of antisense inhibition on translation can be monitored using techniques that include but are not limited to antibody-mediated tests such as RlAs and ELISA, functional assays, or radiolabeling.
  • the extended cDNAs described herein may also be used in gene therapy approaches based on intracellular triple helix formation.
  • Triple helix oligonucleotides are used to inhibit transcription from a genome. They are particularly useful for studying alterations in cell activity as it is associated with a particular gene.
  • the extended cDNAs for genomic DNAs obtainable therefrom) or more preferably, a portion of those sequences can be used to inhibit gene expression in individuals having diseases associated with expression of a particular gene.
  • a portion of the extended cDNA (or genomic DNA obtainable therefrom) can be used to study the effect of inhibiting transcription of a particular gene within a cell.
  • homopurine sequences were considered the most useful for triple helix strategies.
  • homopyrimidine sequences can also inhibit gene expression.
  • Such homopyrimidine oligonucleotides bind to the major groove at homopurine:homopyrimidine sequences.
  • both types of sequences from the extended cDNA or from the gene corresponding to the extended cDNA are contemplated.
  • the sequences of the extended cDNAs are scanned to identify 10-mer to 20-mer homopyrimidine or homopurine stretches which could be used in triple-helix based strategies for inhibiting gene expression.
  • their efficiency in inhibiting gene expression is assessed by introducing varying amounts of oligonucleotides containing the candidate sequences into tissue culture cells which normally express the target gene.
  • the oligonucleotides may be prepared on an oligonucleotide synthesizer or they may be purchased commercially from a company specializing in custom oligonucleotide synthesis, such as GENSET. Paris, France.
  • oligonucleotides may be introduced into the cells using a variety of methods known to those skilled in the art, including but not limited to calcium phosphate precipitation. DEAE-Dextran, electroporation, liposome-mediated transfection or native uptake.
  • Treated cells are monitored for altered cell function or reduced gene expression using techniques such as Northern blotting, RNase protection assays, or PCR based strategies to monitor the transcription levels of the target gene in cells which have been treated with the oligonucleotide.
  • the cell functions to be monitored are predicted based upon the homologies of the target gene corresponding to the extended cDNA from which the oligonucleotide was derived with known gene sequences that have been associated with a particular function.
  • the cell functions can also be predicted based on the presence of abnormal physiologies within cells derived from individuals with a particular inherited disease, particularly when the extended cDNA is associated with the disease using techniques described in Example 53.
  • oligonucleotides which are effective in inhibiting gene expression in tissue culture cells may then be introduced in vivo using the techniques described above and in Example 59 at a dosage calculated based on the in vitro results, as described in Example 59.
  • the natural (beta) anomers of the oligonucleotide units can be replaced with alpha anomers to render the oligonucleotide more resistant to nucleases.
  • an intercalating agent such as ethidium bromide, or the like, can be attached to the 3' end of the alpha oligonucleotide to stabilize the triple helix.
  • the extended cDNAs described herein may also be used to express an encoded protein in a host organism to produce a beneficial effect.
  • the encoded protein may be transiently expressed in the host organism or stably expressed in the host organism.
  • the encoded protein may have any of the activities described above.
  • the encoded protein may be a protein which the host organism lacks or, alternatively, the encoded protein may augment the existing levels of the protein in the host organism.
  • a full length extended cDNA encoding the signal peptide and the mature protein, or an extended cDNA encoding only the mature protein is introduced into the host organism.
  • the extended cDNA may be introduced into the host organism using a variety of techniques known to those of skill in the art.
  • the extended cDNA may be injected into the host organism as naked DNA such that the encoded protein is expressed in the host organism, thereby producing a beneficial effect.
  • the extended cDNA may be cloned into an expression vector downstream of a promoter which is active in the host organism.
  • the expression vector may be any of the expression vectors designed for use in gene therapy, including viral or retroviral vectors.
  • the expression vector may be directly introduced into the host organism such that the encoded protein is expressed in the host organism to produce a beneficial effect.
  • the expression vector may be introduced into cells in vitro. Cells containing the expression vector are thereafter selected and introduced into the host organism, where they express the encoded protein to produce a beneficial effect.
  • the short core hydrophobic region (h) of signal peptides encoded by the 5'ESTS or extended cDNAs derived from the 5'ESTs described herein may also be used as a carrier to import a peptide or a protein of interest, so-called cargo, into tissue culture cells ( Lin et al., J. Biol. Chem., 270: 14225-14258 (1995 ): Du et al., J. Peptide Res., 51: 235-243 (1998 ); Rojas et al., Nature Biotech., 16: 370-375 (1998 )).
  • nucleic acids can be genetically engineered, using techniques familiar to those skilled in the art, in order to link the extended cDNA sequence encoding the h region to the 5' or the 3' end of a DNA sequence coding for a cargo polypeptide.
  • Such genetically engineered nucleic acids are then translated either in vitro or in vivo after transfection into appropriate cells, using conventional techniques to produce the resulting cell permeable polypeptide. Suitable hosts cells are then simply incubated with the cell permeable polypeptide which is then translocated across the membrane.
  • This method may be applied to study diverse intracellular functions and cellular processes. For instance, it has been used to probe functionally relevant domains of intracellular proteins and to examine protein-protein interactions involved in signal transduction pathways (Lin et al., supra : Lin et al., J. Biol. Chem., 271: 5305-5308 (1996 ); Rojas et al., J. Biol. Chem., 271: 27456-27461 (1996 ); Liu et al., Proc. Natl. Acad. Sci. USA. 93: 11819-11824 (1996 ); Rojas et al., Bioch. Biophys. Res. Commun., 234: 675-680 (1997 )).
  • Such techniques may be used in cellular therapy to import proteins producing therapeutic effects. For instance, cells isolated from a patient may be treated with imported therapeutic proteins and then re-introduced into the host organism.
  • oligonucleotides may be antisense oligonucleotides or oligonucleotides designed to form triple helixes, as described in examples 59 and 60 respectively, in order to inhibit processing and maturation of a target cellular RNA.
  • sequences presented in SEQ ID NOs: 47, 73, 79, 89, 91, 96, 126, 128, 134, and 139 are apparently unlikely to be genuine full length cDNAs. These clones are missing a stop codon and are thus more probably 3' truncated cDNA sequences.
  • sequences presented in SEQ ID NOs: 45, 50, 54, 57, 73, 74, 89, 92, 95, 98, 126, 129, 130, 131 and 139 may also not be genuine full length cDNAs based on homology studies with existing protein sequences. Although both of these sequences encode a potential start methionine each could represent a 5' truncated cDNA.
  • SEQ ID NO: 115 was found to be an alternatively spliced transcript and the identities of some of the bases in SEQ ID NO: 131 were corrected.
  • new open reading frames were chosen in some instances.
  • Table IV provides the location of the full coding sequence in SEO ID NO: 45 (i.e. the nucleotides encoding both the signal peptide and the mature protein, listed under the heading FCS location in Table IV), the locations of the nucleotides in SEQ ID NO: 45 which encode the signal peptides (listed under the heading SigPep Location in Table IV), the locations of the nucleotides in SEQ ID NO: 45 which encode the mature proteins generated by cleavage of the signal peptides (listed under the heading Mature Polypeptide Location in Table IV), the locations in SEQ ID NO: 45 of stop codons (listed under the heading Stop Codon Location in Table IV) the locations in SEQ ID NO: 45 of polyA signals (listed under the heading g PolyA Signal Location in Table IV) and the locations of polyA sites (listed under the heading PolyA Site Location in Table IV).
  • Table V lists the sequence identification numbers of the polypeptide of SEQ ID NO: 146, the locations of the amino acid residues of SEQ ID NO: 146 in the full length polypeptide (second column), the locations of the amino acid residues of SEQ ID NO: 146 in the signal peptides (third column), and the locations of the amino acid residues of SEQ ID NO: 146 in the mature polypeptide created by cleaving the signal peptide from the fall length polypeptide (fourth column).
  • the first amino acid of the mature protein resulting from cleavage of the signal peptide is designated as amino acid number 1 and the first amino acid of the signal peptide is designated with the appropriate negative number, in accordance with the regulations governing sequence listings.
  • the first category contains proteins exhibiting more than 70% identical amino acid residues on the whole length of the matched protein. They are clearly close homologues which most probably have the same function or a very similar function as the matched protein.
  • the second category contains proteins exhibiting more remote homologies (40 to 70% over the whole protein) indicating that the protein may have functions similar to those of the homologous protein.
  • the third category contains proteins exhibiting homology (90 to 100%) to a domain of a known protein indicating that the matched protein and the protein of the invention may share similar features.
  • amino acid number 1 the numbering of amino acids in the protein sequences discussed in ( Figures 10 to 15) the first methionine encountered is designated as amino acid number 1.
  • amino acid number 1 the first amino acid of the mature protein resulting from cleavage of the signal peptide is designated as amino acid number 1
  • amino acid number 2 the first amino acid of the signal peptide is designated with the appropriate negative number, in accordance with the regulations governing sequence listings.
  • the protein SEQ ID NO: 146 encoded by the extended cDNA SEQ ID NO: 45 shows homology to human apolipoprotein L ( Genbank accession number AFO 19225 ).
  • the matched protein is a secreted high density lipoprotein associated with apoA-I-containing lipoproteins which play a key role in reverse cholesterol transport.
  • this protein may play a role in lipid metabolism.
  • this protein may be useful in diagnosing and/or treating several types of disorders including, but not limited to, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disorders such as, coronary heart disease, and neurodegenerative disorders such as Alzheimer's disease or dementia.
  • the extended cDNAs described herein or portions thereof can be used for various purposes.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states): as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions: to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination for expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.
  • the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993 )) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
  • proteins or polypeptides provided herein can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening: to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
  • the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
  • Polynucleotides and proteins can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such causes the protein or polynucleotide can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of can be added to the medium in or on which the microorganism is cultured.

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Claims (11)

  1. Polypeptide purifié ou isolé comprenant une séquence étant au moins identique à 80% au polypeptide de Séquence n° 146.
  2. Polypeptide selon la revendication 1, en quoi ledit polypeptide comprend la Séquence n° 146.
  3. Polypeptide selon la revendication 2, en quoi ledit polypeptide est constitué de la Séquence n° 146.
  4. Acide nucléique purifié ou isolé codant le polypeptide de la revendication 2 ou 3.
  5. Acide nucléique selon la revendication 4, en quoi ledit acide nucléique comprend la Séquence n° 45.
  6. Vecteur d'expression comprenant l'acide nucléique de la revendication 4 ou 5.
  7. Cellule hôte contenant un acide nucléique recombiné de la revendication 4 ou 5.
  8. Procédé de fabrication du polypeptide de l'une quelconque des revendication 1 à 3 comprenant les étapes consistant à:
    a. obtenir un ADNc codant ledit polypeptide ;
    b. insérer ledit ADNc dans un vecteur d'expression de sorte que ledit ADNc soit lié opérationnellement à un promoteur ;
    c. et introduire ledit vecteur d'expression dans une cellule hôte ce par quoi ladite cellule hôte produit ledit polypeptide.
  9. Procédé selon la revendication 8 comprenant en outre l'étape d'isolement dudit polypeptide.
  10. Procédé selon la revendication 9 comprenant en outre l'étape de purification dudit polypeptide à l'aide de techniques d'immunochromatographie.
  11. Anticorps purifié ou isolé capable de se lier spécifiquement au polypeptide de l'une quelconque des revendications 1 à 3.
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ATE440951T1 (de) 2009-09-15
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